- Email: [email protected]
Rheological evaluation of gelatin gels prepared with a citric acid derivative as a novel cross-linker
Materials Science and Engineering C 24 (2004) 787 – 790 www.elsevier.com/locate/msec
Rheological evaluation of gelatin gels prepared with a citric acid derivative as a novel cross-linker Hirokatsu Aokia, Tetsushi Taguchib,*, Hirofumi Saitoa, Hisatoshi Kobayashib, Kazunori Kataokab,c, Junzo Tanakab a Furuuchi Chemical Corporation, 6-17-17 Minami-oi, Shinagawa-ku, Tokyo 140-0013, Japan Biomaterials Center, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan c Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113-8656, Japan b
Received 9 June 2004; received in revised form 13 July 2004; accepted 11 August 2004 Available online 2 October 2004
Abstract We developed a tissue adhesive consisting of biomolecules and a citric acid derivative (CAD) with active ester groups. To apply it biomedically, however, gelation time and gel strength must be different depending on the organ and injury. In this study, we added ionic salts or organic solvent to adhesive precursor solutions to modify adhesive properties, such as hardening period and strength. We measured gelation time and mechanical strength of gels under different conditions using a rheometer. We found that adding divalent anions and dimethyl sulfoxide (DMSO) shortened gelation time and increased mechanical strength. D 2004 Elsevier B.V. All rights reserved. Keywords: Gel strength; Citric acid derivative; Dimethyl sulfoxide
1. Introduction Surgical adhesives, such as cyanoacrylate [1] and gelatinresorcinol-formaldehyde [2], developed for clinical use have disadvantages of poor biodegradability and high toxicity. Fibrin glue, often substituted for these adhesives because it originated from human blood, poses the risk of viral infection. To overcome such disadvantages, we developed a tissue adhesive composed of biomolecules and a citric acid derivative (CAD), obtained by esterifying citric acid carboxyl groups with N-hydroxysuccinimide [3], that has the advantages of lower toxicity and biodegradability superior to those of synthetic adhesives. Applying this adhesive biomedically, however, requires that we be able to control physical properties, such as hardening period and mechanical strength. One simple way
* Corresponding author. Tel.: +81 29 851 3354x4498; fax: +81 29 860 4714. E-mail address: [email protected] (T. Taguchi). 0928-4931/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2004.08.021
of modifying these physical properties is to add ionic salts to change the interaction among macromolecules; that is, the existence of ions affects the interaction between ionic functional groups in macromolecules. The crystallization or concentration of biomolecules, such as proteins, is, for example, promoted by adding Hofmeister ions [4]. The solubility of lysozyme, a model protein, is mainly affected by anions [5]. Adding anions at a high concentration changes lysozyme protein molecule interaction from repulsive to attractive, and the order of the Hofmeister ion anion series is maintained. Anion bridges reportedly form between polar functional groups of molecules when salt is added [6]. Such salt-induced variations in intermolecular potential and biomolecule association are thus expected to modify adhesive properties. Many proteins are dissolved in some species of polar organic solvent, such as dimethyl sulfoxide (DMSO). If these organic solvents are replaced by water, the dielectric constant of the solution changes [7]. Ion pairs are generated between oppositely charged ionic functional groups in a solution with a low dielectric constant. DMSO has a lower
788
H. Aoki et al. / Materials Science and Engineering C 24 (2004) 787–790
was measured at a gap of 1.0 mm and a load shear stress of 1.0 Pa.
3. Results and discussion
Fig. 1. Variations in gelation time due to the addition of inorganic salts. o: NaCl; 4: NaNO3; 5: Na2SO4; R : PBS.
dielectric constant (e r=46.6) than water (e r=78.54) at 298 K, so the formation of ion pairs is promoted by using DMSO in place of water, which results in attraction between biomolecules. Polar organic solvents can thus be used to change the properties of adhesives. We clarified the effect of ionic salts and organic solvent in an adhesive solution on adhesive properties by measuring viscoelasticity.
2. Experiments 2.1. Materials Gelatin (pI=8–9) extracted from porcine skin by an acid process was purchased from Sigma-Aldrich Japan (Tokyo, Japan). Special-grade sodium chloride, sodium nitrate, sodium sulfate, disodium hydrogenphosphate, sodium dihydrogenphosphate dehydrate, and DMSO were purchased from Wako Pure Chemical Industries, (Osaka, Japan). CAD was synthesized by esterifying citric acid with N-hydroxysuccinimide as reported elsewhere [3].
Fig. 1 shows gelation time t gel of the adhesive when different inorganic salts are added, i.e., sodium chloride, sodium nitrate, sodium sulfate, and phosphate buffer solution (PBS). The pH of solutions was 5–5.5 for sodium chloride, sodium nitrate, and sodium sulfate and 6.0 for PBS. With the addition of the monovalent anion, t gel increased with increasing salt concentration, but for the divalent anion, t gel decreased with increasing salt concentration. Gelation time varied with the anion species, corresponding to the Hofmeister series of anions, which indicates the salting out of proteins. Gel strength variation supports the salting-out effect, with the order of gel strength varying with the anion species consistent with the Hofmeister series (Fig. 2). The aggregation of biomolecules is promoted by Hofmeister ions, which dehydrate proteins, hardening the gel. The order of effectiveness of anions in salting out hydrophobic groups, such as benzyl groups (nonpolar molecules), for example, is SO 42NOH , FNClNNO3NClO4NI [9]. Divalent ions are generally more effective than monovalent ions in salting out, so the effectiveness of PBS, which consists of HPO42 and H2PO4, may rank next to SO42. Biomolecules consist of nonpolar groups and negative or positive functional groups, so Hofmeister ions also interact with peptide bonds in biomolecules through a nonspecific ion–dipole interaction [10] because the peptide bond has a significant dipole moment. Such interaction contributes to salting in of biomolecules. The result of salt-concentration dependence for gel strength of the gelatin-CAD adhesive for monovalent anions implies that the effect of salting in exceeded that of salting out, while the reverse was true for divalent anions.
2.2. Gelation time and gel strength measurement Gelation time and gel strength were measuring using a Haake RS1 rheometer (Thermo Electron, Dreieich, Germany). The gelatin concentration was fixed at 20 wt.% and the CAD concentration at 100 mM. Ionic salts and DMSO were added to the gelatin solution. To measure gelation time, the gelatin solution was heated to 50 8C, then CAD was added at time t=0. The mixture was then stirred vigorously and centrifuged to remove air bubbles. Samples were then introduced between measurement plates at 37 8C. Gelation time was defined as the crossing point of GV=GW with time variation [8]. Gel disks 20 mm in diameter and 1.5 mm thick were used to measure gel strength. Viscoelasticity
Fig. 2. Variations in gel strength due to the addition of inorganic salts. o: NaCl; 4: NaNO3; 5: Na2SO4; R : PBS.
H. Aoki et al. / Materials Science and Engineering C 24 (2004) 787–790
Fig. 3. Gel strength of adhesive with a water/DMSO mixed solution.
Regarding the addition of DMSO, gel strength increased with increasing DMSO concentration (Fig. 3). The dielectric constant of the water/DMSO mixture was smaller at a high DMSO concentration. More ion pairs exist between oppositely charged functional groups due to electrical attraction at that side. Gel strength increases monotonously with increasing DMSO concentration. The increase in gel strength with increasing DMSO concentration is thus a consequence of electrical attraction between charged functional groups. The swelling ratio decreased with increasing DMSO concentration (Fig. 4). Active ester groups of CAD react competitively with amino groups of gelatin and of water, so gel cross-linking density increases at higher DMSO concentrations. The low swelling ratio of gels prepared at a lower DMSO concentration was due to the complete reaction of active ester groups with amino groups of gelatin. t gel of the adhesive was minimal near a 30 wt.% DMSO concentration in a water/DMSO mixed solvent (Fig. 5). In cross-linking, active ester groups are first hydrolyzed, so water content is required to promote cross-
789
Fig. 5. Gelation time for gelatin gels with a water/DMSO mixed solution. o: no CAD; R : 100 mM of CAD.
linking. As discussed above, adding DMSO mainly affects the electrostatic attraction between oppositely charged biomolecules. At a DMSO concentration lower than 40 mM, electrostatic attraction shortens gelation time. DMSO, however, interferes with the formation of the helical gelatin structure during cooling at higher DMSO concentrations because DMSO molecules interact with amide through hydrogen bonding. Gelation time thus increases at DMSO concentrations higher than 40 mM. The gelation time of gelatin with 100 mM of CAD is longer than that with 0 mM of CAD because excess CAD also interferes with the formation of the helical structure of gelatin.
4. Conclusions Gelatin adhesive with a CAD cross-linker is hardened by adding divalent anions due to electrostatic attraction and salting out. Divalent anions help shorten gelation time t gel of the adhesive. Gel strength increases with increasing DMSO concentration with the addition of DMSO. Some water content shortens t gel, which changes the hydrolysis of CAD. The minimum gelatin gel hardening time (2 min) was obtained in a water/DMSO mixed solvent with a DMSO concentration of 30 wt.%.
Acknowledgement
Fig. 4. Swelling ratio of gelatin gels in a phosphate buffer solution.
This work was financially supported in part by a Grantin-Aid for Young Scientists (B) (No. 15700344) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan and by Industrial Technology Research Grant Program in ’04 from New Energy and Industrial Technology Development Organization (NEDO) of Japan.
790
H. Aoki et al. / Materials Science and Engineering C 24 (2004) 787–790
References [1] Y.-C. Tseng, S.-H. Hyon, Y. Ikada, Y. Shimizu, K. Tamura, S. Hitomi, J. Appl. Biomater. 1 (1990) 111. [2] N.S. Braunwald, W.J. Gay, C. Tatooles, Surgery 59 (1966) 1024. [3] T. Taguchi, H. Saito et al., (submitted for publication) Mater. Sci. Eng., C, Biomim. Mater., Sens. Syst. [4] F. Hofmeister, Arch. Exp. Path. Pharmakol. 24 (1888) 247.
[5] A. Ducruix, J.P. Guilloreau, M. Ries-Kautt, A. Tardieu, J. Cryst. Growth 168 (1996). [6] D.T. Bowron, J.L. Finney, Phys. Rev. Lett. 89 (2002) 215508. [7] E. Tomilla, A. Pajunen, Suom. Kemistil., B 41 (1969) 172. [8] C.Y.M. Tung, P.J. Dynes, J. Appl. Polym. Sci. 27 (1982) 569. [9] W.F. McDevit, F.A. Long, J. Am. Chem. Soc. 74 (1952) 1733. [10] A. Hamabata, P.H. von Hippel, Biochemistry 12 (1973) 1264.
Rheological evaluation of gelatin gels prepared with a citric acid derivative as a novel cross-linker Hirokatsu Aokia, Tetsushi Taguchib,*, Hirofumi Saitoa, Hisatoshi Kobayashib, Kazunori Kataokab,c, Junzo Tanakab a Furuuchi Chemical Corporation, 6-17-17 Minami-oi, Shinagawa-ku, Tokyo 140-0013, Japan Biomaterials Center, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan c Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113-8656, Japan b
Received 9 June 2004; received in revised form 13 July 2004; accepted 11 August 2004 Available online 2 October 2004
Abstract We developed a tissue adhesive consisting of biomolecules and a citric acid derivative (CAD) with active ester groups. To apply it biomedically, however, gelation time and gel strength must be different depending on the organ and injury. In this study, we added ionic salts or organic solvent to adhesive precursor solutions to modify adhesive properties, such as hardening period and strength. We measured gelation time and mechanical strength of gels under different conditions using a rheometer. We found that adding divalent anions and dimethyl sulfoxide (DMSO) shortened gelation time and increased mechanical strength. D 2004 Elsevier B.V. All rights reserved. Keywords: Gel strength; Citric acid derivative; Dimethyl sulfoxide
1. Introduction Surgical adhesives, such as cyanoacrylate [1] and gelatinresorcinol-formaldehyde [2], developed for clinical use have disadvantages of poor biodegradability and high toxicity. Fibrin glue, often substituted for these adhesives because it originated from human blood, poses the risk of viral infection. To overcome such disadvantages, we developed a tissue adhesive composed of biomolecules and a citric acid derivative (CAD), obtained by esterifying citric acid carboxyl groups with N-hydroxysuccinimide [3], that has the advantages of lower toxicity and biodegradability superior to those of synthetic adhesives. Applying this adhesive biomedically, however, requires that we be able to control physical properties, such as hardening period and mechanical strength. One simple way
* Corresponding author. Tel.: +81 29 851 3354x4498; fax: +81 29 860 4714. E-mail address: [email protected] (T. Taguchi). 0928-4931/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2004.08.021
of modifying these physical properties is to add ionic salts to change the interaction among macromolecules; that is, the existence of ions affects the interaction between ionic functional groups in macromolecules. The crystallization or concentration of biomolecules, such as proteins, is, for example, promoted by adding Hofmeister ions [4]. The solubility of lysozyme, a model protein, is mainly affected by anions [5]. Adding anions at a high concentration changes lysozyme protein molecule interaction from repulsive to attractive, and the order of the Hofmeister ion anion series is maintained. Anion bridges reportedly form between polar functional groups of molecules when salt is added [6]. Such salt-induced variations in intermolecular potential and biomolecule association are thus expected to modify adhesive properties. Many proteins are dissolved in some species of polar organic solvent, such as dimethyl sulfoxide (DMSO). If these organic solvents are replaced by water, the dielectric constant of the solution changes [7]. Ion pairs are generated between oppositely charged ionic functional groups in a solution with a low dielectric constant. DMSO has a lower
788
H. Aoki et al. / Materials Science and Engineering C 24 (2004) 787–790
was measured at a gap of 1.0 mm and a load shear stress of 1.0 Pa.
3. Results and discussion
Fig. 1. Variations in gelation time due to the addition of inorganic salts. o: NaCl; 4: NaNO3; 5: Na2SO4; R : PBS.
dielectric constant (e r=46.6) than water (e r=78.54) at 298 K, so the formation of ion pairs is promoted by using DMSO in place of water, which results in attraction between biomolecules. Polar organic solvents can thus be used to change the properties of adhesives. We clarified the effect of ionic salts and organic solvent in an adhesive solution on adhesive properties by measuring viscoelasticity.
2. Experiments 2.1. Materials Gelatin (pI=8–9) extracted from porcine skin by an acid process was purchased from Sigma-Aldrich Japan (Tokyo, Japan). Special-grade sodium chloride, sodium nitrate, sodium sulfate, disodium hydrogenphosphate, sodium dihydrogenphosphate dehydrate, and DMSO were purchased from Wako Pure Chemical Industries, (Osaka, Japan). CAD was synthesized by esterifying citric acid with N-hydroxysuccinimide as reported elsewhere [3].
Fig. 1 shows gelation time t gel of the adhesive when different inorganic salts are added, i.e., sodium chloride, sodium nitrate, sodium sulfate, and phosphate buffer solution (PBS). The pH of solutions was 5–5.5 for sodium chloride, sodium nitrate, and sodium sulfate and 6.0 for PBS. With the addition of the monovalent anion, t gel increased with increasing salt concentration, but for the divalent anion, t gel decreased with increasing salt concentration. Gelation time varied with the anion species, corresponding to the Hofmeister series of anions, which indicates the salting out of proteins. Gel strength variation supports the salting-out effect, with the order of gel strength varying with the anion species consistent with the Hofmeister series (Fig. 2). The aggregation of biomolecules is promoted by Hofmeister ions, which dehydrate proteins, hardening the gel. The order of effectiveness of anions in salting out hydrophobic groups, such as benzyl groups (nonpolar molecules), for example, is SO 42NOH , FNClNNO3NClO4NI [9]. Divalent ions are generally more effective than monovalent ions in salting out, so the effectiveness of PBS, which consists of HPO42 and H2PO4, may rank next to SO42. Biomolecules consist of nonpolar groups and negative or positive functional groups, so Hofmeister ions also interact with peptide bonds in biomolecules through a nonspecific ion–dipole interaction [10] because the peptide bond has a significant dipole moment. Such interaction contributes to salting in of biomolecules. The result of salt-concentration dependence for gel strength of the gelatin-CAD adhesive for monovalent anions implies that the effect of salting in exceeded that of salting out, while the reverse was true for divalent anions.
2.2. Gelation time and gel strength measurement Gelation time and gel strength were measuring using a Haake RS1 rheometer (Thermo Electron, Dreieich, Germany). The gelatin concentration was fixed at 20 wt.% and the CAD concentration at 100 mM. Ionic salts and DMSO were added to the gelatin solution. To measure gelation time, the gelatin solution was heated to 50 8C, then CAD was added at time t=0. The mixture was then stirred vigorously and centrifuged to remove air bubbles. Samples were then introduced between measurement plates at 37 8C. Gelation time was defined as the crossing point of GV=GW with time variation [8]. Gel disks 20 mm in diameter and 1.5 mm thick were used to measure gel strength. Viscoelasticity
Fig. 2. Variations in gel strength due to the addition of inorganic salts. o: NaCl; 4: NaNO3; 5: Na2SO4; R : PBS.
H. Aoki et al. / Materials Science and Engineering C 24 (2004) 787–790
Fig. 3. Gel strength of adhesive with a water/DMSO mixed solution.
Regarding the addition of DMSO, gel strength increased with increasing DMSO concentration (Fig. 3). The dielectric constant of the water/DMSO mixture was smaller at a high DMSO concentration. More ion pairs exist between oppositely charged functional groups due to electrical attraction at that side. Gel strength increases monotonously with increasing DMSO concentration. The increase in gel strength with increasing DMSO concentration is thus a consequence of electrical attraction between charged functional groups. The swelling ratio decreased with increasing DMSO concentration (Fig. 4). Active ester groups of CAD react competitively with amino groups of gelatin and of water, so gel cross-linking density increases at higher DMSO concentrations. The low swelling ratio of gels prepared at a lower DMSO concentration was due to the complete reaction of active ester groups with amino groups of gelatin. t gel of the adhesive was minimal near a 30 wt.% DMSO concentration in a water/DMSO mixed solvent (Fig. 5). In cross-linking, active ester groups are first hydrolyzed, so water content is required to promote cross-
789
Fig. 5. Gelation time for gelatin gels with a water/DMSO mixed solution. o: no CAD; R : 100 mM of CAD.
linking. As discussed above, adding DMSO mainly affects the electrostatic attraction between oppositely charged biomolecules. At a DMSO concentration lower than 40 mM, electrostatic attraction shortens gelation time. DMSO, however, interferes with the formation of the helical gelatin structure during cooling at higher DMSO concentrations because DMSO molecules interact with amide through hydrogen bonding. Gelation time thus increases at DMSO concentrations higher than 40 mM. The gelation time of gelatin with 100 mM of CAD is longer than that with 0 mM of CAD because excess CAD also interferes with the formation of the helical structure of gelatin.
4. Conclusions Gelatin adhesive with a CAD cross-linker is hardened by adding divalent anions due to electrostatic attraction and salting out. Divalent anions help shorten gelation time t gel of the adhesive. Gel strength increases with increasing DMSO concentration with the addition of DMSO. Some water content shortens t gel, which changes the hydrolysis of CAD. The minimum gelatin gel hardening time (2 min) was obtained in a water/DMSO mixed solvent with a DMSO concentration of 30 wt.%.
Acknowledgement
Fig. 4. Swelling ratio of gelatin gels in a phosphate buffer solution.
This work was financially supported in part by a Grantin-Aid for Young Scientists (B) (No. 15700344) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan and by Industrial Technology Research Grant Program in ’04 from New Energy and Industrial Technology Development Organization (NEDO) of Japan.
790
H. Aoki et al. / Materials Science and Engineering C 24 (2004) 787–790
References [1] Y.-C. Tseng, S.-H. Hyon, Y. Ikada, Y. Shimizu, K. Tamura, S. Hitomi, J. Appl. Biomater. 1 (1990) 111. [2] N.S. Braunwald, W.J. Gay, C. Tatooles, Surgery 59 (1966) 1024. [3] T. Taguchi, H. Saito et al., (submitted for publication) Mater. Sci. Eng., C, Biomim. Mater., Sens. Syst. [4] F. Hofmeister, Arch. Exp. Path. Pharmakol. 24 (1888) 247.
[5] A. Ducruix, J.P. Guilloreau, M. Ries-Kautt, A. Tardieu, J. Cryst. Growth 168 (1996). [6] D.T. Bowron, J.L. Finney, Phys. Rev. Lett. 89 (2002) 215508. [7] E. Tomilla, A. Pajunen, Suom. Kemistil., B 41 (1969) 172. [8] C.Y.M. Tung, P.J. Dynes, J. Appl. Polym. Sci. 27 (1982) 569. [9] W.F. McDevit, F.A. Long, J. Am. Chem. Soc. 74 (1952) 1733. [10] A. Hamabata, P.H. von Hippel, Biochemistry 12 (1973) 1264.