Mechanism and kinetics of the crosslinking ... - Wiley Online Library

Mechanism and Kinetics of the Crosslinking Reaction between Biopolymers Containing Primary Amine Groups and Genipin MICHAEL F. BUTLER, YIU-FAI NG, PAUL D. A. PUDNEY Unilever R&D Colworth, Sharnbrook, Bedfordshire, MK44 1LQ, United Kingdom

Received 27 June 2003; accepted 14 August 2003

ABSTRACT: The reaction mechanism of chitosan, bovine serum albumin (BSA), and gelatin with genipin (a natural crosslinking reagent) was examined with infrared, ultraviolet–visible, and 13C NMR spectroscopies; protein-transfer reaction mass spectrometry; photon correlation spectroscopy; and dynamic oscillatory rheometry. Two reactions that proceeded at different rates led to the formation of crosslinks between primary amine groups. The fastest reaction to occur was a nucleophilic attack on genipin by a primary amine group that led to the formation of a heterocyclic compound of genipin linked to the glucosamine residue in chitosan and the basic residues in BSA and gelatin. The second, slower, reaction was the nucleophilic substitution of the ester group possessed by genipin to form a secondary amide link with chitosan, BSA, or gelatin. A decreased crosslinking rate in the presence of deuterium oxide rather than water suggested that acid catalysis was necessary for one or both of the reactions to proceed. The behavior of the gel time with polymer concentration was consistent with second-order gelation kinetics resulting from an irreversible crosslinking process, but was complicated by the oxygen radical-induced polymerization of genipin that caused the gels to assume a blue color in the presence of air. The lower elastic modulus attained after a given time during crosslinking of the globular protein BSA as compared to the coiled protein gelatin, despite possessing more crosslinkable basic residues, demonstrated the importance of protein secondary and tertiary structures in determining the availability of sites for crosslinking with genipin in protein systems. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3941–3953, 2003

Keywords: chitosan; bovine serum albumin; gelatin; genipin; covalent crosslinking; hydrogels; biopolymers; crosslinking

INTRODUCTION Chitosan is the partially deacetylated from of chitin [poly(N-acetyl-D-glucosamine)], which is a structural polysaccharide found in insects, crustacea, and some fungi.1 Its component residues, glucosamine and acetyl-D-glucosamine, are shown in Figure 1(a,b), respectively. As a commercially available natural cationic polyelectrolyte that swells at

Correspondence to: M. F. Butler (E-mail: michael.butler@ unilever.com) Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 41, 3941–3953 (2003) © 2003 Wiley Periodicals, Inc.

low pH, with good biocompatibility and low cytotoxicity, it has recently attracted much interest as a stimulus-responsive, mucoadhesive material1–11 for the controlled delivery of active molecules in pharmaceutical applications. In the area of foods, chitosan is considered to be a dietary fiber with several beneficial health properties.12 These include anticholesterolaemic, antiulcer, and antiuricemic properties13 stemming from its ability to bind fatty acids, bile acids, phospholipids, and uric acid. Chitosan dissolves readily in acidic conditions (below ca. pH 6.5) to form a viscous solution. In the presence of phosphate ions, thermogels can be formed.14 –16 Covalently crosslinked hydrogels 3941

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BUTLER, NG, AND PUDNEY

Figure 1. Chemical formulas for (a) glucosamine, (b) acetyl-glucosamine, (c) genipin, and (d) geniposide, where glu represents glucose.

may also be formed after reaction with various chemicals such as glutaraldehyde, diglycidyl ethers, epoxides,3,4 and ␤-cyclodextrin.17 These reagents are relatively cytotoxic. An alternative natural crosslinking reagent does exist, however, called genipin. It has recently provoked interest18 –29 for its ability to crosslink chitosan and certain proteins containing residues with primary amine groups, particularly, gelatin and soya protein isolates. Genipin is obtained from its parent compound, geniposide, via enzymatic hydrolysis with ␤-glucosidase. The formulas for genipin and geniposide are depicted in Figure 1(c,d), respectively. Geniposide is isolated from the fruits of Genipa americana and Gardenia jasminoides Ellis and constitutes about 4 – 6% of dried fruit. Genipa americana is found in tropical America, from Mexico and the Caribbean to Argentina, where it is widely planted for its shade and fruit. The fruits, with a taste similar to quinces, are eaten raw, used to make a sour, refreshing drink, cooked with sugar to flavor liquor, and used as a diuretic and as a remedy for respiratory ailments. Gardenia jasminoides plants are grown in the Far East, and their fruits have long been used in Chinese medicine for their antiphlogistic, anti-inflammatory, diuretic, choleretic, and haemostatic properties. Extracts from the fruits have also been used to form brilliant blue pigments via the reaction of genipin with primary amines in the presence of oxygen,30 –35 which are used as a food dye commonly known as gardenia blue. These blue pigments are of particular importance because they are highly stable to heat, pH, and light.35

Initial observation of the formation of dimers of genipin in the presence of glycine led to the suggestion that genipin could be used to covalently crosslink proteins containing residues with primary amine groups.18 Subsequently, it has been used in studies of tissue fixation to crosslink collagen and gelatin,21,25 in foodstuff to crosslink soy protein isolates,28,29 and in studies of drug delivery, where it has been used to crosslink chitosan.18 –20,22–24,26,27 In all of these applications, genipin was chosen because of its markedly lower cytotoxicity as compared with alternative crosslinkers such as glutaraldehyde.20,21,24,27 Physiological studies have shown that geniposide, which is highly abundant in certain edible fruits, is converted into genipin in the gastrointestinal tract, with no adverse effects.36,37 The aim of this work was to extend the understanding of the reaction mechanism between chitosan, as an example of a biopolymer containing primary amine groups, and genipin by combining measurements of the gelation kinetics with those of the reaction mechanism. Other biopolymers, namely, bovine serum albumin (BSA) and gelatin, that possess basic residues (e.g., lysine or arginine) with primary amine groups were also examined to extend the relevance of the findings from the study of chitosan and genipin.

EXPERIMENTAL Materials and Sample Preparation Chitosan was supplied by Primex and was 90% deacetylated. Glucosamine and acetyl-glu-

CROSSLINKING REACTION

cosamine were obtained from Sigma Chemical Co. and were reagent grade. Genipin was supplied by Challenge Bioproducts, Ltd. (Taiwan). Gelatin (porcine, bloom 175) and BSA were obtained from Sigma Chemical. Two percent (weight/volume) stock solutions of chitosan, glucosamine, and acetyl-glucosamine were prepared by dissolving the powder in 1% (v/v) acetic acid. Genipin solutions of the required concentration were prepared by dissolving the genipin powder in 1% (v/v) acetic acid for reaction with chitosan and in deionized, distilled water for the BSA and gelatin solutions. To test for the effect of isotopic subsitution, a genipin solution was made in 1% (v/v) acetic acid with deuterium oxide as the solvent rather than water. Twenty percent (weight/volume) gelatin and BSA solutions were prepared by dissolving the powder in deionized, distilled water. The gelatin was dissolved at 60 °C, and the BSA was dissolved at room temperature. For all experiments, 15 mL of the chitosan, glucosamine, acetyl-glucosamine, gelatin, or BSA stock solution were pipetted into a glass vial and mixed with 5 mL of the required genipin solution to produce a sample with a final chitosan, glucosamine, or acetyl-glucosamine concentration of 1.5% (w/v), a final gelatin or BSA concentration of 15% (w/v), and a genipin concentration of 1, 5, 10, 25, or 100 mM. Experiments were also conducted by reacting 10 mM of genipin with amidated pectin, low-methoxy pectin, skim milk powder, sodium caseinate, whey protein, soy isolate, maltodextrin, gum arabic, and glutamine at 40 °C. Ultraviolet–Visible (UV–Vis) Spectroscopy UV–vis spectroscopy experiments were performed with a PerkinElmer Lambda40 spectrophotometer on the individual components and on chitosan, glucosamine, and acetyl-glucosamine samples containing an overall genipin concentration of 1 mM. The samples were placed in a quartz cell with a path length of 5 mm. A layer of mineral oil was placed on top of the sample, and the cell was sealed with a plastic lid to limit solvent evaporation and oxygen ingress during the kinetic runs. Experiments were performed at 20 °C. Spectra were obtained every 20 min over a period of 48 –96 h in the wavelength range from 900 to 220 nm, at 2 nm resolution. Fourier Transform Infrared (FTIR) Spectroscopy FTIR spectra were performed with a Bio-Rad FTS6000 FTIR spectrometer equipped with a di-

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amond attenuated total reflectance accessory (Golden Gate, Specac, Ltd.). The solutions of chitosan (1.5% w/v), genipin (10 mM), and chitosan (1.5% w/v) containing an overall genipin concentration of 10 mM were placed onto the diamond surface and covered with a small Petri dish, which was sealed with Vaseline petroleum jelly to limit evaporation of water from the sample during the experiment. Spectra were obtained every minute over a period of 24 h. 13

C NMR Spectroscopy

13

C NMR spectra were acquired with a Bruker AMX400 spectrometer at 100 MHz with 4260 scans and a relaxing delay of 1 s. The spectra were thus acquired every 2 h and 31 min. The sample was maintained at a temperature of about 2.5 °C during the course of the experiment. Line broadening of 30 Hz was used to process the spectra. ACD/CNMR software (version 3.5) was used to generate a predicted spectrum for genipin to assist with the interpretation of the experimental results.

Protein-Transfer Reaction-Mass Spectrometry (PTRMS) PTR-MS experiments were performed on chitosan, glucosamine, and acetyl-glucosamine in the presence of genipin (10 mM) at 20 °C. The genipin was introduced after approximately 30 min to ascertain the response of the chitosan, glucosamine, and acetyl-glucosamine solutions without crosslinker. The experiments were performed over a time period of about 12 h, and volatile organic compounds with a molecular weight range from 30 to 150 were detected.

Photon Correlation Spectroscopy (PCS) PCS was used to measure the change in molecular size during the reaction of BSA and genipin. Measurements were made on a mixture containing 15 wt % BSA and 25 mM of genipin with a Malvern Instruments Autosizer 4700 equipped with a Uniphase Cyonics argon ion laser operating at a wavelength of 488 nm and power output of 20 mW. Measurements of the scattering intensity fluctuations were made at a scattering angle of 90°. Particle sizes were obtained from the measured field correlation function with the Contin analysis method.

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Rheology The development of the oscillatory rheological behavior of chitosan, gelatin, and BSA solutions containing genipin was characterized with a Couette geometry in a dynamic stress rheometer supplied by Rheometric. A sample volume of about 20 mL was used, and a layer of mineral oil was placed on top of the sample to minimize solvent evaporation and oxygen ingress during the course of the experiments, which lasted between 48 and 96 h. The autostress facility was used to ensure that the correct stress was applied throughout the crosslinking reaction to yield accurate and reliable values of the modulus, which changed by several orders of magnitude as the sample changed from a relatively nonviscous liquid to a stiff gel. The rheological behavior of chitosan solutions (1.5% w/v) and chitosan– genipin gels was thoroughly characterized before the collection of the kinetic data. An initial stress of 0.025 Pa was applied to the sample, rising to 2 Pa when a strong gel had formed after several days. The storage and loss moduli were measured at an applied frequency of 0.1 Hz, and data were collected every minute. To investigate the effect of crosslinker concentration on the chitosan– genipin crosslinking kinetics, samples with genipin concentrations of 1, 5, 10, 25, and 100 mM were examined at a fixed temperature (20 °C). To investigate the effect of temperature, samples containing 10 mM of genipin were analyzed at 20, 40, 60, and 80 °C. The rheology of gelatin and BSA samples with a protein concentration of 15% (w/v) reacting with 10 mM of genipin was measured at a temperature of 40 °C, where both proteins were still in solution and not aggregated.

RESULTS Observations Glucosamine, acetyl-glucosamine, and genipin solutions were initially clear and colorless, and the chitosan, BSA, and gelatin solutions were initially clear and slightly yellow. After a time period that was dependent on the temperature and amount of genipin, for concentrations exceeding 0.1 mM, the solutions containing glucosamine or chitosan with genipin first became green, then blue. At very low genipin concentrations (0.01 mM), glucosamine solutions became clear and

Figure 2. UV spectra of glucosamine, acetyl-glucosamine, chitosan, and genipin.

slightly yellow, whereas the chitosan solutions were not discernibly altered in appearance. The solution containing acetyl-glucosamine and genipin remained clear and colorless, even after several weeks at room temperature or after heating to 80 °C for several hours. No qualitative differences were observed between mixtures of chitosan and genipin (at sufficient concentrations to form a gel) that were left in the dark and those that were left in daylight or artificial light. The exposure of the mixture to air had a significant influence on the development of the blue color, however. In all of the chitosan gels, the blue coloration was deepest near the surface exposed to air, and gradually moved further into the sample. The BSA and gelatin gels became uniformly blue, however. In the presence of deuterium oxide, the crosslinking reaction between chitosan and genipin was much slower than when the solvent was water. The mixture of chitosan and genipin in deuterium oxide did not form a gel or develop any blue coloration until several days had elapsed, in contrast to several hours in the presence of water. BSA and gelatin went blue when mixed with solutions containing all concentrations of genipin, but took longer and required larger quantities of polymer and genipin to form gels. BSA and gelatin solutions with polymer concentrations in excess of about 10 wt % and genipin concentrations in excess of about 15 mM were required to form a gel. UV–Vis Spectroscopy The UV–vis spectra of glucosamine, acetyl-glucosamine, chitosan, and genipin are depicted in Figure 2. Glucosamine possessed major absorp-


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Figure 3. Evolution of the UV–vis spectrum of glucosamine after mixing with genipin, with selected regions of the spectrum expanded.

Figure 4. Evolution of the UV–vis spectrum of acetylglucosamine after mixing with genipin, with selected regions of the spectrum expanded.

tion peaks at 240 and 280 nm, and acetyl-glucosamine possessed a UV absorption peak at 275 nm. The spectrum of chitosan was a superposition of the contributions from glucosamine and acetylglucosamine, but was dominated by the spectrum of glucosamine. Genipin possessed a major UV absorption at 240 nm. The development of the UV spectra of glucosamine, acetyl-glucosamine, and chitosan mixed with genipin is illustrated in Figures 3–5, respectively. For the spectrum of the glucosamine– genipin mixture, the intensity of the genipin peak at 240 nm decreased slightly at the start of the reaction before reaching a constant value. After a certain time, a new peak appeared at 605 nm that continued to increase in intensity throughout the course of the reaction. For the acetyl-glucosamine/ genipin mixture, there was also an initial decrease in the intensity of the genipin peak at 240 nm [Fig. 4(b)]. However, no new peak appeared around 605 nm. The results for chitosan mixed with genipin were partially similar to those for glucosamine, in that an initial decrease in the 240-nm genipin peak was measured as well as the delayed appearance and subsequent continual increase in intensity of the peak at 605 nm. However, the chitosan– genipin

mixture additionally displayed an increase in the intensity of the peaks at 240 and 280 nm from the start of the reaction.

Figure 5. Evolution of the UV–vis spectrum of chitosan after mixing with genipin.

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Figure 6. Evolution of the FTIR spectrum of the chitosan– genipin mixture, with decreasing absorbance of the peaks at 1550 and 1414 cm⫺1 and increasing absorbance of the peak at 1092 cm⫺1 indicated by arrows.

FTIR Spectroscopy IR spectra measured at different times during the reaction between chitosan and genipin are displayed in Figure 6. Significant absorbance was observed at 1710, 1550, 1414, 1280, 1155, 1092, 1076, and 1022 cm⫺1. The peaks at 1022 and 1155 cm⫺1 were assigned to vibrational modes associated with saccharide units in chitosan, and the peak at 1280 cm⫺1 was due to the chitosan hydroxyl group.22,23 The peaks at 1076 and 1092 cm⫺1 resulted from C–O and/or C–N stretching.22 The band at 1414 cm⫺1 was assigned to a ring stretching mode in the genipin molecule.22 The peaks at 1550 and 1710 cm⫺1 were due to protonated amine groups on the chitosan molecule22,23 and carboxylic acid groups in the acetic acid solvent, respectively. The absorbance peak at 1092 cm⫺1 gradually increased in intensity from the onset of addition of genipin to the chitosan solution, accompanied by a slower increase in intensity of the peak at 1076 cm⫺1. Significant decreases in absorbance were also measured for the peaks at 1550 and 1414 cm⫺1, and a new peak appeared at about 1630 cm⫺1 toward the later stages of the reaction (apparent after 7920 s but not at 3960 s in Fig. 6). No changes were measured in the peaks at 1700, 1300, 1150, and 1050 cm⫺1. Figure 7 shows that the increase in intensity of the 1076-cm⫺1 peak was directly proportional to that of the 1092-cm⫺1 peak. 13

C NMR Spectroscopy

Experimentally measured and predicted 13C NMR spectra of genipin are portrayed in Figure

Figure 7. Change in the ratio of FTIR absorbances 1092:1076 cm⫺1 after mixing chitosan and genipin.

8(a,b), respectively. There was agreement between the positions and relative intensities of the peaks in the experimental and predicted spectra.

Figure 8. (a) Predicted 13C NMR spectrum for genipin, showing the peak assignments for the carbon atoms marked in Figure 1. (c) and (b) experimentally measured 13C NMR spectrum for genipin dissolved in 1% w/v acetic acid.


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the rate of decrease of the intensity became greater toward the later stages of the experiment. PTR-MS

Figure 9. Experimentally measured 13C NMR spectra for (a) chitosan and (b) chitosan– genipin mixture.

The peaks in the experimental genipin spectrum in Figure 8, and therefore some of the peaks in the chitosan– genipin mixture, were thereby assigned to the carbon atoms in the genipin molecule, as numbered in Figure 1. The spectra for chitosan and a chitosan– genipin mixture are shown in Figure 9, with the assigned genipin peaks marked. The spectrum of the chitosan– genipin mixture was a combination of the spectra from the individual components. The change in intensity of each of these peaks was measured, and plots of the peak intensity versus time for selected peaks are shown in Figure 10. The intensity of the peak at 155 ppm, from the C3 carbon atom on the unreacted genipin molecule, decreased from the start of the reaction between chitosan and genipin. No change in intensity was measured for the peak at 172 ppm from the C11 carbon atom on the unreacted genipin molecule. Decreases in intensity were measured for the peaks at 112, 36, 130, and 62 ppm from the genipin C4, C6, C7, and C10 carbon atoms, respectively. For these four carbon atoms,

RH⫹ species with masses of 61 and 43, which were identified as signatures from the acetic acid present in the solvent,38 were detected as soon as the glucosamine and acetyl-glucosamine solutions were placed in the reaction vessel, and reached a constant concentration that was maintained throughout the experiment. No immediate changes were detected upon addition of the genipin solution to either sample, but for the solution containing glucosamine, shown in Figure 11(a), an RH⫹ species with a mass of 33 was detected after about 150 min. This species was interpreted as methanol.38 The concentration of this species then increased throughout the remainder of the experiment. For the solution containing acetylglucosamine, also shown in Figure 11(a), the RH⫹ species with a mass of 33 was not detected at any stage of the experiment. Similarly, a delayed production of the RH⫹ species with a mass of 33 (methanol) was detected in the chitosan sample, shown in Figure 11(b), several minutes after the addition of genipin. The time at which this occurred coincided with the time at which a significant increase in elastic modulus was detected [marked by the arrow in Fig. 11(b)] during rheological measurements on a separate chitosan– genipin sample reacted under the same conditions. PCS At the start of the experiment, PCS detected a single particle diameter at about 7.5 nm for BSA. During the course of the experiment, and shown in Figure 12, the particle diameter continually increased. After about 3 h, the signal was too weak to obtain a reliable particle size because of the significant amount of absorption that occurred as a result of the change in color of the sample. Measurements were not made on the chitosan– genipin system because unambiguous interpretation of the correlation function for the chitosan solutions could not be made. Rheology The evolution of the elastic modulus (G⬘) with time after mixing chitosan with genipin is displayed in Figure 13 for chitosan– genipin mixtures with a genipin concentration of 10 mM that was reacted at different temperatures. An initial

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Figure 10. Change in peak height with time after mixing the 13C NMR peaks from significant genipin carbon atoms in a chitosan– genipin mixture [see Fig. 1(c) for assignment]: (a) C11, (b) C3, (c) C4, (d) C6, (e) C7, and (f) C10.

induction time, during which no increase in the value of the G⬘ was observed, was followed by a sharp increase in the G⬘. Although the G⬘ did not reach a plateau within the timescale of the experiment, the values of the G⬘ measured at the different temperatures appeared to be asymptotically heading toward similar values (of the order of 1000 Pa). Increasing the reaction temperature decreased the induction time before significant increases in the gel strength were measured. A logarithmic plot of the inverse of the gel time, measured as the time at which the value of the G⬘ exceeded those of the loss modulus (G⬙) versus the

inverse of the absolute temperature is shown in Figure 14. An apparent Arrhenius relationship was observed. The evolution of the G⬘ with time after mixing is portrayed in Figure 15 for chitosan– genipin mixtures reacting at 40 °C and containing different concentrations of genipin. With the exception of the sample containing 1 mM of genipin, the G⬘ did not reach a plateau. Increasing the genipin concentration decreased the induction time during which no increase in the value of the G⬘ was measured and enabled higher gel strengths to be reached in a given time after mixing. The value of the G⬘ for the


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Figure 13. Evolution of the elastic modulus for for chitosan (1.5% w/v)/genipin (10 mM) mixtures reacting at different temperatures.

Figure 11. PTR-MS measurements for (a) glucosamine and acetyl-glucosamine mixed with genipin and (b) chitosan mixed with genipin.

sample containing 100 mM of genipin had reached 10,000 Pa and was still increasing at the same (power law) rate after 48 h, when measurements

Figure 12. PCS measurements of BSA molecular diameter after mixing with genipin.

ceased. Likewise, the sample containing 25 mM of genipin possessed an G⬘ in excess of 1000 Pa after the same time that showed no indication of a decrease in its (power-law) rate of increase. The samples containing lower concentrations of genipin showed signs of tending toward a plateau G⬘, however. Figure 16 is a double logarithmic plot of the inverse of the gel time against genipin concentration. A straight line was obtained, indicating an inverse power-law relationship. Figure 17 shows the cure curves of BSA and gelatin reacting with 10 mM of genipin at 40 °C, which exhibited similar behavior to those of chitosan. The increase in G⬘ was less rapid for the protein samples, and the modulus at a given time after mixing was lower than that achieved for chitosan reacting with genipin under the same conditions.

Figure 14. Arrhenius plot for the inverse of the gel time for 10 mM of genipin reacting with chitosan at different temperatures.

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Figure 15. Evolution of the elastic modulus for chitosan (1.5% w/v)/genipin mixtures reacting at 40 °C for different genipin concentrations.

DISCUSSION Previous studies20,22–24,26 have demonstrated that genipin reacts with materials containing primary amine groups, such as chitosan and some peptides and polypeptides, to form covalently crosslinked networks. It is believed that the crosslinks are formed via two reactions involving different sites on the genipin molecule. From previous FTIR and 13C NMR studies of chitosan– genipin mixtures,22 the two reactions involving genipin that are believed to result in crosslinking of polymers containing primary amine groups are shown in Figure 18. Reaction scheme 1 in Figure 18 is an SN2 nucleophilic substitution reaction that involves the replacement of the ester group on the genipin

Figure 16. Variation of the inverse of the gel time with genipin concentration at 40 °C for chitosan– genipin mixtures.

Figure 17. Evolution of the elastic modulus for gelatin (15% w/v)/genipin (10 mM) and BSA (15% w/v)/ genipin (10 mM) mixtures.

molecule by a secondary amide linkage. In this study, its occurrence was identified by the evolution of methanol that was detected in chitosan– genipin and glucosamine– genipin mixtures with PTR-MS. Because methanol was only evolved for glucosamine and chitosan, but not for acetyl-glucosamine, we can conclude that only the glucosamine residues in chitosan undergo this reaction. The nucleophile was, therefore, the primary amine group. Additional evidence for the conversion of primary amine groups on chitosan to secondary amide linkages between chitosan and genipin was provided by the decrease in absorbance of the protonated amine IR peak at 1550 cm⫺1 combined with the appearance of the secondary amide absorbtion at 1630 cm⫺1 at the later stages of the reaction.23 Because the G⬘ began to increase at the same time as the first traces of methanol were detected, we can conclude that the nucleophilic subsitution of the ester group on the genipin molecule was the second reaction of the crosslinking process. The constant intensity of the 13C NMR peak due to the C11 carbon atom in the genipin molecule, which is the carbon atom linked to the ester group, provided further evidence that nucleophilic subsitution of the ester group occurred during the later stages of the crosslinking reaction. If it were the first stage, then a change in intensity from the onset of the reaction would have occurred. The other half of the crosslink, which must have already formed by the time that the ester substitution occurred, is believed to form via reaction scheme 2 in Figure 18. This reaction begins with an initial nucleophilic attack of the genipin C3 carbon atom from a primary amine group to


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Figure 18. Crosslinking reactions involving genipin.

form an intermediate aldehyde group. Opening of the dihydropyran ring is then followed by attack on the resulting aldehyde group by the secondary amine formed in the first step of the reaction. A heterocyclic compound of genipin linked to the glucosamine residue in chitosan and the residues containing primary amine groups in BSA and gelatin, via the primary amine group, is thereby formed. In this study, the immediate increase in the intensity of an IR band at 1092 cm⫺1 at the expense of the 1076-cm⫺1 band, combined with the decrease in intensity of the protonated amine band at 1550 cm⫺1 upon mixing chitosan and genipin, is interpreted as the formation of C–N bonds at the expense of C–O bonds during the formation of the heterocyclic genipin– chitosan compound. Because this occurred as soon as the genipin and chitosan were mixed, this must have been the first reaction to occur. Additional evidence for the immediate occurrence of this reaction was provided by the immediate decrease in intensity of the 13C NMR peak that was attributed to the C3 carbon atom in the genipin molecule and the immediate increase in intensity of the UV–vis absorption at 280 nm that is believed to be due to the presence of a heterocyclic genipin– glucosamine/chitosan compound.22 The necessity for acid catalysis of at least the SN2 ester substitution reaction and possibly the ring-opening reaction39 explains the much slower rate of

crosslinking in the presence of deuterium oxide. The constant absorbance of the IR peaks at 1710, 1280, 1155, and 1022 cm⫺1 justifies their assignment to bonds present in the chitosan or acetic acid solvent that did not participate in the crosslinking reaction. The formation of blue pigments suggests that, in addition to the reactions involved in crosslinking, other more complex reactions occurred. Previous studies of the blue pigments obtained in the reaction of genipin with amino acids found that they were formed from the oxygen radical-induced polymerization of genipin and dehydrogenation of intermediate compounds, following the ring-opening reaction because of attack of genipin by a primary amine group.22,34,40,41 The change in intensity of the 13C NMR peaks due to the genipin C6, C8, and C10 carbon atoms that were measured during the reaction provided evidence for the occurrence of reactions other than those involving the C3 and C11 carbon atoms that were directly involved in crosslinking. These results support the suggestion that the polymerization reactions are induced by the presence of oxygen radicals because the blue coloration was initially more pronounced at the interface of the gelled samples and gradually moved down through the sample with time. These results also suggest that the polymerization reactions could only occur once one of the crosslinking reactions had taken

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place. No blue pigments formed when genipin was mixed with acetyl-glucosamine because it was unable to initiate either of the crosslinking reactions.39 Moreover, the continual increase in BSA particle size measured by PCS may be interpreted as the polymerization of genipin attached to BSA molecules. If the increase in particle size was due to BSA molecule crosslinking, then it would be expected to occur at a later time once the ester substitution reaction occurred, and furthermore to occur in units of the unreacted BSA molecule size. From this interpretation, polymerization was therefore induced by the formation of the heterocyclic genipin compound rather than the ester substitution reaction. Crosslinking of chitosan molecules by genipin polymers of different lengths can explain the sustained increase in G⬘ and the high G⬘ that were achieved for the higher genipin concentrations in the chitosan– genipin system. If crosslinks were formed from single genipin molecules, gelation would eventually be slowed because of steric constraints on the number of genipin molecules that could link separate network polymer molecules. Crosslinks formed from several genipin molecules will be more flexible, allowing network polymer molecules to be linked that are further apart. Analysis of rheologically obtained gel time may be performed. In the simplest interpretation, the reciprocal of the gel time, 1/tc, is predicted to be related to the temperature, T, and the concentration of the components, C, by the following equation:42

冉冊

1 Ea ⬁C m⫺1 exp tc RT

(1)

where m is the order of the crosslinking reaction, and Ea is the activation energy of the rate-limiting step in the crosslinking process. From these results for chitosan, at fixed concentration an Arrhenius relationship was observed. The value of Ea that was measured from this plot was 39 kJ mol⫺1. This value must relate to the rate-limiting step in the formation of the gel, which is the nucleophilic substitution of the ester group on the genipin molecule by the secondary amide linkage between chitosan and genipin. At fixed temperature, m ⫺ 1 was measured to be 0.78, which gave a value of m of 1.78. This value was close to 2, which is the value expected from a simple irreversible gelation process. The occurrence of the various polymerization reactions that gave the blue color to the gels may be the reason for the deviation from second-order reaction kinetics.

The lower values of G⬘ that were attained at a given time for BSA and gelatin as compared to chitosan, along with the higher polypeptide concentrations required to form gels, was due to the lower number of amine groups available for participating in the crosslinking reaction in these systems. For a globular protein such as BSA, the secondary and tertiary structures are also important because it may be assumed that the lysine or arginine residues involved in the crosslinking reaction must be near the outside surface of the molecule for them to be effective. Gelatin possesses a coiled conformation at the temperature at which it was crosslinked. The greater availability of the crosslinkable residues in gelatin as compared to those in BSA, even though BSA possesses a greater number of basic residues,43– 46 may therefore explain the higher modulus obtained in the crosslinked gelatin sample as compared with BSA.

CONCLUSIONS The reaction mechanism between biopolymers containing primary amine groups (chitosan, BSA, and gelatin) and genipin was investigated with IR, UV–vis, and 13C NMR spectroscopies; PTRMS, PCS, and dynamic oscillatory rheometry. Two separate reactions led to the formation of crosslinks between primary amine groups. The fastest, and therefore first, reaction to occur was a nucleophilic attack of the genipin C3 carbon atom from a primary amine group that led to the formation of a heterocyclic compound of genipin linked to the glucosamine residue in chitosan and the basic residues in BSA and gelatin. The slowest second reaction was the nucleophilic substitution of the ester group possessed by genipin to release methanol and form a secondary amide link with chitosan, BSA, or gelatin. A decreased reaction rate in the presence of deuterium oxide rather than water suggested that acid catalysis was necessary for one or both of the reactions to proceed. The behavior of the gel time with polymer concentration was consistent with second-order gelation kinetics resulting from an irreversible crosslinking process. It was, however, complicated by the oxygen radical-induced polymerization of genipin that occurred once the heterocyclic genipin compund linked to chitosan, BSA, or gelatin had formed and caused the gels to assume a blue color in the presence of air. Protein secondary and tertiary structures were important in determining the availability of sites for


crosslinking in protein systems because a lower G⬘ was attained after a given time during crosslinking of the globular protein BSA than the coiled protein gelatin, despite possessing more crosslinkable basic residues. Alan Peilow, of Unilever R&D Colworth, is thanked for his assistance with the PTR-MS measurements. Don Farrer, retired, formerly of Unilever R&D Colworth, is gratefully acknowledged for his many useful discussions.

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