Novel Chitosan-Based Films Cross-Linked by Genipin with Improved

Biomacromolecules 2004, 5, 162-168

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Novel Chitosan-Based Films Cross-Linked by Genipin with Improved Physical Properties J. Jin, M. Song,* and D. J. Hourston Institute of Polymer Technology and Material Engineering, Loughborough University, Loughborough LE11 3TU, United Kingdom Received August 7, 2003; Revised Manuscript Received November 6, 2003

Novel cross-linked chitosan-based films were prepared using the solution casting technique. A naturally occurring and nontoxic cross-linking agent, genipin, was used to form the chitosan and chitosan/poly(ethylene oxide) (PEO) blend networks, where two types of PEO were used, one with a molecular weight of 20 000 g/mol (HPEO) and the other of 600 g/mol (LPEO). Genipin is used in traditional Chinese medicine and extracted from gardenia fruit. Importantly, it overcomes the problem of physiological toxicity inherent in the use of some common synthetic chemicals as cross-linking agents. The mechanical properties and the stability in water of cross-linked and un-crosslinked chitosan and chitosan/PEO blend films were investigated. It was shown that, compared to the transparent yellow, un-cross-linked chitosan/PEO blend films, the genipincross-linked chitosan-based film, blue in color, was more elastic, was more stable, and had better mechanical properties. Genipin-cross-linking produced chitosan networks that were insoluble in acidic and alkaline solutions but were able to swell in these aqueous media. The swelling characteristics of the films exhibit sensitivity to the environmental pH and temperature. The surface properties of the films were also examined by contact angle measurements using water and mixtures of water/ethanol. The results showed that, with the one exception of cross-linked pure chitosan in 100% water, the cross-linked chitosan and chitosan/PEO blends were more hydrophobic than un-crosslinked ones. Introduction Chitosan, a natural biomaterial, has recently attracted much attention from scientists in different parts of the world.1-3 It has been reexamined and found to be a useful resource as a functional material.4,5 Because of the presence of amino groups, chitosan is soluble in aqueous acidic media and forms viscous solutions that can be used to produce gels in various forms, e.g., beads, membranes, coatings, fibers, and sponges.6-8 The amino and hydroxyl groups of chitosan give rise to it being easily chemically modified. Chitosan, as a functional material, offers a special set of characteristics: biocompatibility, biodegradability, and anti-bacterial properties.6,7 It is also biologically inert, safe for human use, and stable in the natural environment.5,9,10 The above characteristics make chitosan suitable for use in a number of biomedical applications, including artificial skin, tissue regeneration, and drug delivery systems.2,11-13 It is vital that in all of these applications the material must be demonstrably nontoxic. In other words, the polymer, its possible degradation products, any residual monomer, and all additives must be free of harmful effects. These requirements have not always been met. Chitosan for tissue regeneration and similar medical applications must be biocompatible and mechanically satisfactory. In terms of mechanical performance, for example in structural materials in hip reconstruction and replacement, the biomaterial’s strength and other physical properties are * To whom correspondence should be addressed.

critically important and the most difficult to satisfy.1,2 As a result, the application of chitosan as a biomedical material has been limited to a few specific areas,1,2 which include wound dressings, sutures, and skin and tissue engineering. Many recent attempts14-17 have tried to improve the mechanical properties of chitosan. Modification of chitosan through blending with other polymers18,19 and cross-linking20,21 are both convenient and effective in improving its physical properties for practical applications. There have been some reports22-24 dealing with polyblends of chitosan with other natural or synthetic polymers. Recently, chitosan and poly(ethylene oxide) (PEO) blends have been reported for the preparation of membranes for haemodialysis25 and semiIPNs for pH-sensitive drug delivery.26 PEO is a biologically inert and flexible polymer. It has suitable uses for medical purposes, and it can improve the mechanical properties of blends. These properties of chitosan/PEO films were examined by Alexeev et al.,14 and it was observed that for the chitosan/PEO blends containing 16.7 wt % of PEO there was a six times improvement in the elongation at break and a doubling in the tensile strength. It was shown that at 16.7 wt % of PEO the film structure is likely to be homogeneous, and therefore, this gives rise to the improved mechanical characteristics.14 The mechanical properties and the stability of chitosan/PEO blends have also been investigated by Wang et al.15 Their results showed that there were specific interactions between PEO and chitosan and that the maximum interaction between them occurred at 20wt % PEO content. The results also indicated that these chitosan blend

10.1021/bm034286m CCC: $27.50 © 2004 American Chemical Society Published on Web 12/09/2003

Chitosan-Based Films Cross-Linked by Genipin

Biomacromolecules, Vol. 5, No. 1, 2004 163

Scheme 1. Schematic of Genipin Chemical Structure

of PEO in different weight percentage ratios and the mixtures were stirred overnight at room temperature. The degassed and well-mixed solutions were then cast onto glass plates, evaporated at 35°C and dried to constant weight. The thickness of the dried films ranged from 0.3 to 0.6 mm. Preparation of Genipin-Cross-Linked Films. 0.5 wt % of genipin (chitosan/genipin ) 100/0.5 by weight) was dissolved in 3 mL of water and then was added to the chitosan solutions, or to the chitosan/PEO mixed solutions, under stirring for 30 min at room temperature. After 2 h, the solutions started to turn light blue and became increasingly viscous. The solutions were then immediately cast on glass plates and dried to constant weight at ambient temperature. The cross-linked chitosan and chitosan/PEO blends become dark blue after 1 day. The thicknesses of the dried films were measured to be 0.3-0.6 mm. Mechanical Testing. Tensile strength and percentage elongation were measured on film strips using a L1oyd Instruments (U.K.) tensometer operated according to the D638 ASTM standard. The tensometer was fitted with a 500N load cell. The extension rate was 10 mm/min. To examine the physical properties of the films as a function of moisture content, they were placed in a relative humidity chamber over distilled water (pH 7). The moisture content was determined by drying to constant weight. Each test was repeated at least 3 times and the average is reported. Solubility and Stability in Neutral Water. The crosslinked/un-crosslinked chitosan and chitosan/LPEO blend films were immersed in 100 mL of methanol for 24 h to remove the acetic acid in the films and were then taken out and dried for 12 h at 35 °C. The dried films were weighed accurately. The dried films were then immersed in distilled water (pH 7) for 8 h. They were then taken out and dried for 24 h under vacuum at 35°C. A second weighing was conducted to determine the amount of LPEO extracted from the blends. All of the films used were from the same samples as used for the tensile strength measurements. Stability in water, therefore, can be expressed by the following equation:

films were unstable, for the films partly dissolved in distilled water after having been immersed for 8 h. These blend films are, therefore, limited in their uses and applications. Many other investigations20,21 have been carried out to overcome such undesirable properties by the method of crosslinking. The most common synthetic chemical used as a cross-linking reagent is gluteraldehyde.20 Among others are formaldehyde,27 epoxy compounds,28 and dialdehyde starch.29 However, all of these cross-linking agents are chemically synthesized and are not free from the problems caused by physiological toxicity.30,31 Genipin is found in traditional Chinese medicine and extracted from gardenia fruit.32,33 It has been reported that genipin, whose chemical structure is shown in Scheme 1, is an effective naturally occurring-cross-linking agent and can react spontaneously with amino acids or proteins to form dark blue pigments.34 Sung and co-workers35-38 have undertaken studies to investigate the cytotoxicity, feasibility, and biocompatibility of genipin for tissue fixation. The results of these studies38 demonstrated that genipin was 10 000 times less cytotoxic than glutaraldehyde, a finding that encouraged us to investigate the use of this agent to produce novel materials for medical purposes. The goal of the current work is to develop novel chitosan/ PEO blend films, designed to be mechanically resilient, chemically inert, and stable, by using genipin as the crosslinker to prepare the chitosan networks. The aim of this paper is to report on the mechanical properties and the stability in water of these novel films. Experimental Section Materials. Chitosan was purchased from Fluka, U.K. The molecular weight of chitosan was measured by means of gel permeation chromatography (GPC). Mw ) 3.0 × 105, Mn ) 5.8 × 104, and Mw/Mn ) 5.5. The degree of deacetylation of the chitosan was 88%, which was determined by Fourier transform infrared (FT-IR). Poly(ethylene oxide) with molecular weight of 20 000 g/mol (HPEO) was obtained from BDH Laboratory Supplies, U.K. Poly(ethylene glycol) powder with viscosity average molecular weight of 600 g/mol (LPEO) was obtained from the Aldrich Chemicals, U.K. Genipin was obtained from Challenge Bioproducts Co., Taiwan. Acetic acid was obtained from Aldrich Chemicals, U.K. All chemicals were used without further purification. Preparation of Chitosan /PEO Blend Films. Chitosan was dissolved in 1 wt % aqueous acetic acid at roomtemperature overnight to obtain a concentration of 1.5 wt/ v%. The viscous chitosan solution was filtered through filter paper to remove any undissolved gel. The clear, light yellow chitosan solution was then mixed with a 5% aqueous solution

S ) W2/W1 × 100 where S is the percentage of the films remaining after immersion in neutral water. W1 and W2 are the weight of dried films before and after immersion in water, respectively. Each experiment was repeated three times, and the average value is reported. Swelling Studies. The swelling characteristics of the crosslinked and the un-crosslinked chitosan and their blend films were determined by swelling the films in media of different pHs at both room temperature and 38°C. The samples (approximately 0.05 g) were first treated as described above to remove the residual acetic acid. Blends and networks were immersed in buffer solutions ranging from pH 2 to 13. The immersion time was 48 h. It was confirmed that after a 48-h period the films had reached their swelling equilibrium. The films were withdrawn from the buffer solutions, and their wet weights were determined after first blotting with a filter paper to remove surface absorbed water, followed immediately by weighing. The specific solution content, or

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weight uptake percentage, of the films is expressed by the following equation: Esw ) [(Ws - Wd)/Wd] × 100 where Esw is the percentage water adsorption of the film at equilibrium. Wd and Ws are the weights of the samples in the dry and swollen states, respectively. Each swelling experiment was repeated three times, and the average values are reported. Contact Angle Analysis. Contact angle measurements were carried out using a Data Physics OCA20 instrument, fitted with an automatic image capture system. The liquids used were pure water, mixtures of 70% water and 30% ethanol, and 50% water and 50% ethanol. Because of the rapid evaporation of ethanol and the need to maintain consistency of results, dynamic tracking was used to capture the contact angle of the drop every second for up to 1 min. All films were neutralized with NaOH before use. Results and Discussion Tensile Strength and Percentage Elongation. The tensile strength and the elongation at break plotted against the %LPEO in the dry films of both cross-linked/un-crosslinked chitosan and chitosan-LPEO blends are shown in parts a and b of Figure 1, respectively. It was observed that the film prepared from pure chitosan was rather brittle. The highest tensile strength was found in the blend with 20 wt % of LPEO, and the greatest enhancement of elongation at the break for the blend films occurred at 65% LPEO content, the highpoint of a general trend of elongation increase as the LPEO content increased. Cross-linking with naturally occurring genipin gave rise to improvements in both mechanical properties: the tensile strength and the elongation at break for LPEO systems. Table 1 shows the results of tensile strength and percentage elongation for HPEO systems. It is clearly seen that crosslinking with naturally occurring genipin did not give rise to improvements in both mechanical properties: the tensile strength and the elongation at break for the HPEO systems which results from the lower miscibility of chitosan with HPEO.39 Figure 2, parts a and b, presents the stress-strain curves for un-cross-linked and cross-linked chitosan blend films (with 0.5% of genipin) with 50 wt % LPEO content after their conditioning in environments of various relative humidities. It can be seen in both cases that, as the water content in the films increased, the elongation at break increased, whereas the tensile strength decreased. However, cross-linking gave rise to greater increases in both tensile strength and elongation at break than the equivalent dry samples. At 15% water content, the increase in the tensile strength of the cross-linked film was more than twice that of the un-crosslinked one. At 25% water content, the elongation at break of the cross-linked film reached 80% and its tensile strength was found to be 15MPa, and the elongation at break was 35% and its tensile strength was found to be 6 MPa for the un-crosslinked film. Because it is important to prevent the carrier in a drug delivery system

Figure 1. (a) Tensile strength of cross-linked and un-crosslinked dried chitosan/LPEO blends films versus blend composition. (b) Elongation at break of cross-linked and un-crosslinked dried chitosan/ LPEO blend films versus blend composition. Table 1. Tensile Strength and Percentage Elongation for HPEO Systems

PEO (%)

elongation at break (%)

stress at break (MPa)

10 20 30

Un-Cross-Linked HPEO System 19 25 14

60 63 50

10 20 30

Cross-Linked HPEO System 15 17 15

55 70 58

from disintegrating when the water content of its surrounding environment increases, the greater improvements in mechanical properties due to cross-linking are of real significance. Figure 3, parts a and b, shows the stress-strain plots of the cross-linked chitosan blend films (50 wt % LPEO) with different genipin contents at two water contents. Both results indicated that 0.1% of genipin content gave the greatest increase in elongation at break. When the genipin content



Figure 2. (a) Stress of un-crosslinked chitosan-LPEO(50%) blend films versus strain with different water contents. (b) Stress of crosslinked chitosan-LPEO(50%) blend films versus strain with different water contents.

Figure 3. (a) Stress-strain curves for chitosan-LPEO(50%) blend films with a 5% water content and a range of cross-linking content. (b) Stress-strain curves for chitosan-LPEO(50%) blend films with a 13% water content and a range of cross-linking levels.

was increased further, to 0.5% and 0.8%, the improvements in elongation at break were not as significant as that for 0.1% genipin. The reasons for this are not yet clear. In future research, the matter will be investigated further. Chitosan can be described in terms of the degree of deacetylation and average molecular weight in conjunction with their film-forming properties.40 It has been observed that the film formation of un-crosslinked blends is closely related to their composition. Above 50% (w/w) and 65% (w/w) of LPEO content, the blends are phase separated, and no useful films could be formed. Similar results have been reported by Zhao.41 However, cross-linked blends at 80 wt % or above of LPEO overcame this problem. The use of genipin as a cross-linker clearly improves the properties of chitosan-based films. Stability of Cross-Linked Blend Films in Water. Knowing that chitosan is naturally insoluble in water (pH 7), whereas both LPEO and HPEO are soluble, the stability of both cross-linked and un-crosslinked blend films in neutral water was examined. Figure 4, parts a and b, shows the degrees of stability of the films plotted against wt % of LPEO (HPEO). A comparison shows that the un-cross-linked chitosan-LPEO film is more stable than the un-cross-linked chitosan-HPEO film, with the latter being very unstable

which is because the miscibility of LPEO with chitosan is higher than that of HPEO with chitosan.39 Cross-linking, however, produced noticeable increases in the stability of both blends, in particular these of the chitosan/HPEO blends. This effect of cross-linking is of great importance to the biomedical applications of such materials since it is linked to their stability during fabrication and storage and while in service. Swelling of the Cross-Linked Blend Films. Genipincross-linked chitosan networks have the advantages of being insoluble in acidic and alkaline solutions and the ability to swell in these aqueous media. It is clear that chitosan is soluble at pH e 6.5.7 Figure 5a shows that the swellability of pure chitosan films was affected by the genipin content. From the figure, it can be seen that films with low genipin contents (0.1%) dissolved at pH ) 2, whereas films with 0.5% and 0.8% of genipin were insoluble in acidic solution. Furthermore, for insoluble films, an increase in the crosslinker content meant a decrease in the extent of swelling. The degree of swelling of a series of films in different pH environments at 23 and 38 °C is shown in Figure 5, parts b and c, respectively. It can be seen that the swelling behavior of the films was strongly dependent on the pH value of the swelling medium, the wt % of LPEO in the film, and the

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Figure 4. (a) Comparison of the stabilities of cross-linked and uncrosslinked chitosan/HPEO blend films (0.5% genipin). (b) A comparison of the stabilities of cross-linked and un-crosslinked chitosan/ LPEO blend films (0.5% genipin).

temperature. When the temperature was increased from 23 to 38 °C, swelling in pH e 7 buffer solutions increased by 50-100%. In alkaline buffer solutions, however, there were no notable increases in swelling, although the greatest swelling now occurred under the lower alkaline condition of pH 10 rather than the previous pH 12. It can be concluded that genipin-cross-linked chitosan networks resulted in significantly different swelling characteristics in the various pH buffer solutions. The increased swelling of the crosslinked films at pHs lower than 7 may be ascribed to the hydrolysis of amide linkages in the cross-linked chitosan network by acid and the regeneration of amine groups in networks.36 Because of the fact that the amino groups reformed in the network could be protonated in acid, the equilibrium ratio of the swelling of the chitosan film in acid was larger than that in the neutral solution. It would be a desirable characteristic for a controlled-release system with controllable swelling ability to be pH- and temperaturesensitive. These films are, therefore, of general interest for biomedical applications such as artificial muscles or switches, biomedical separation systems, and drug controlled-release systems.1,29 Contact Angle Analysis. The surface properties of the chitosan films and the blends for both cross-linked and un-

Figure 5. (a) Swelling behavior of chitosan films at 23°C with different cross-linking levels. (b) The swelling behavior of cross-linked chitosan/ LPEO blend films at 23°C. (c) The swelling behavior of cross-linked chitosan/LPEO blend films at 38°C.

crosslinked samples were investigated by contact angle analysis. Figure 6 shows the contact angles for these different liquid media when a drop was placed on the surfaces of the pure chitosan film and the network. The greatest contact angles, chitosan and network films, were achieved at 100% water, measuring at 78.5° and 62.6°, respectively. Crosslinking, therefore, resulted in an improvement in the wettability of the chitosan films. However, this improvement was only present with 100% water, because chitosan networks exhibited higher contact angles than the un-crosslinked films



Figure 6. Contact angles of the cross-linked and un-crosslinked chitosan films with 100% water, 70% water/30% ethanol, and 50% water/50% ethanol.

in 70% water/30%ethanol and 50%water/50% ethanol systems. Figure 7a-c shows the contact angles for all of the chitosan/LPEO (HPEO) blends and networks in all three liquid systems. As seen in most cases, the contact angles for cross-linked chitosan and the blends were much higher than for the un-crosslinked ones. The surface became more hydrophobic because of cross-linking, but the angle decreased gradually when 10, 20, and 30 wt % of LPEO (or HPEO) were incorporated. The lower contact angles corresponded well to the fact that the LPEO (or HPEO) has high chain mobility and hydrophilicity. An increase in the LPEO (or HPEO) content, thus, allowed a greater wettability of the surface. A slight, but gradual, decrease in contact angles was also observed when water was diluted with 30 and 50% of ethanol. On the whole, the contact angle analysis was helpful in aiding the understanding of the surface properties of the blends and networks. Although cross-linking resulted in decreases in the wettability of the film surfaces in some cases, this unwanted effect was negated to some extent by the addition of PEO. Furthermore, the swelling ability of cross-linked chitosan and blends in ethanol is of particular importance to its success as a drug carrier. For example, the anti-cancer drug, Taxol, is only soluble in ethanol.42 Conclusions Using naturally occurring genipin, as a cross-linking agent, gave rise to improvements in the mechanical properties and stability in water of chitosan and chitosan blend films and produced interesting results concerning their swelling properties and wettability. The increases in the elongation at break and the tensile strength of the cross-linked films, especially in those with high water content, would prevent them from disintegrating in potential applications. With the use of crosslinking, there were also noticeable increases in the stability of the chitosan/LPEO (or HPEO) blends. The swelling behavior of the films exhibited pH and temperature-dependent characteristics. However, the swelling extent can be reduced by an increase in the genipin content. In terms of wettability, with the one exception of cross-linked pure chitosan in 100% water, the cross-linked chitosan and

Figure 7. (a) Contact angles for the cross-linked and un-crosslinked chitosan/PEO blend films for 100% water. (b) Contact angles for the cross-linked and un-crosslinked chitosan/PEO blend films for 70% water/30% ethanol. (c) Contact angles for the cross-linked and uncrosslinked chitosan/PEO blend films for 50% water/50% ethanol.

chitosan/PEO both molecular weights blends were more hydrophobic than un-crosslinked ones. These novel films are sensitive to the environment and should be investigated further for drug delivery and biomaterials applications. References and Notes (1) Francis, S. J. K.; Matthew, H. W. T. Biomaterials 2000, 21, 2589. (2) Ravi Kumar, N. V. M. React. Funct. Polym. 2000, 46, 1. (3) Sigh, D. K.; Ray, A. R. Macromol. Chem. Phys. 2000, C40, 69.

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Novel Chitosan-Based Films Cross-Linked by Genipin with Improved

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