Biomacromolecules 2004, 5, 1310-1315
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Oligochitosan Derivatives Bearing Electron-Deficient Aromatic Rings for Adsorption of Amitriptyline: Implications for Drug Detoxification Dong-Won Lee and Ronald H. Baney* Department of Materials Science and Engineering, University of Florida, Gainesville, Florida 32611 Received January 30, 2004; Revised Manuscript Received March 15, 2004
The objective of this work is the synthesis of water-soluble oligochitosan derivatives with electron deficient aromatic rings for selective and rapid adsorption of amitriptyline through π-π complexation. Oligochitosan was chemically modified under homogeneous conditions in dimethyl sulfoxide (DMSO). 1H NMR, FT-IR, and MALDI-TOF were employed in characterization, confirming that the electron deficient aromatic rings were chemically attached to the backbone of oligochitosan. Thromboelastography (TEG) revealed functionalized oligochitosan derivatives did not affect blood clotting. 1H NMR was also utilized to observe the aromatic-aromatic interaction between electron deficient aromatic rings on oligochitosan and electron rich aromatic rings in amitriptyline. The chemical shift variation of aromatic protons in oligochitosan derivatives was followed to monitor the aromatic-aromatic interaction. Upfield shift of aromatic protons on benzenesulfonyl and dinitrobenzenesulfonyl groups was observed upon the addition of amitriptyline, supporting the formation of π-π complexes through aromatic-aromatic interactions. Dinitrobenzenesulfonyl rings show a larger variation in chemical shift due to the presence of the electron deficient nitro groups. Introduction Chitosan, a linear copolymer of N-acetyl-D-glucosamine and D-glucosamine linked by glucosidic linkage, is derived from chitin which is the second most abundant natural polymer.1,2 Much attention has been paid to chitosan as a precursor for a value-added biopolymer in biomedical and pharmaceutical areas due to nontoxicity, biological activity, biocompatibility, biodegradability, and potential of physical and chemical modification.3-6 Chitosan has been useful for gene delivery vehicles, wound and burn healing, soft and hard contact lenses, artificial organ membranes, and in the immobilization of enzymes or living cells.7 Chitosan nanoparticles prepared by ionotropic gelation have been extensively utilized as a drug carrier in oral, perenteral, and intravenous administration. Calvo et al.8 reported the formation of the hydrophilic chitosan-poly(ethylene oxide) nanoparticles and the potential application for the administration of the therapeutic molecules. It was shown that the chitosan nanoparticles have a great protein loading capacity and provide a continuous release of the entrapped protein up to 1 week.8 A stable freeze-dried formulation of insulin-loaded chitosan nanoparticles was prepared for nasal administration by ionotropic gelation followed by freeze-drying in the presence of cryoprotective agents.9 These authors proposed that freeze-dried chitosan nanoparticles are useful vehicles for increasing the nasal absorption of insulin. Oligochitosan (DP < ∼10) is prepared by cleaving the main chains with acidic hydrolysis, enzymatic degradation, and irradiation.10,11 Oligochitosan has been reported to be * To whom corresponds should be addressed. Tel: 352-846-3785. Fax: 352-846-3355. E-mail:
[email protected].
soluble in neutral water or organic solvents, more biocompatible, and biodegradable.12,13 The unique chemical and physiological characteristics of oligochitosan led us to use it as a toxic drug scavenger in the blood vessel. To date, no attempt has been reported on oligochitosan for in vivo drug detoxification. Drug detoxification has the concept opposite to the drug delivery. Tricyclic antidepressant, amitriptyline (Figure 1), was chosen as a target drug, because it is the most widely prescribed antidepressant and frequently used for committing suicide.14 Amitriptyline is reported to be extremely hydrophobic and cardiotoxic. One of the strongly desired characteristics for drug detoxification is the selective binding toward target drugs. Hydrophilic chitosan does not have a selective binding capability for tricyclic hydrophobic amitriptyline. Functionalized oligochitosan able to selectively bind amitriptyline could be obtained through the chemical modification. Chemical modification of chitosan has been extensively studied for various purposes, such as enhancement of solubility in water or organic solvents,15,16 introduction of hydrophobicity,17 and development of blood compatibility and stealth property.18-20 However, most chemical reactions have been performed with high molecular weight chitosan under heterogeneous conditions due to their poor solubility in organic solvents. One of the challenges in this work is the homogeneous chemical modification of oligochitosan with π-π complex forming groups. A proposed approach for selective binding toward amitriptyline is based on the assumption that electron deficient aromatic rings chemically bound to oligochitosan not only selectively bind the amitriptyline but also deactivate its toxicity. Aromaticaromatic interaction is commonly observed in biological molecules such as DNA, drugs, and protein and molecular
10.1021/bm049935o CCC: $27.50 © 2004 American Chemical Society Published on Web 05/07/2004
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Figure 1. Chemical structure of amitriptyline and oligochitosan derivatives.
recognition.21,22 Although the magnitude of this attractive interaction is relatively small (2∼5 kcal/mol), the interaction is believed to play an essential role in natural self-assemblies and molecular recognition processes.23 The primary objective of the present work is to synthesize the intravenously injectable functionalized oligochitosan derivatives capable of selectively reducing the free concentration of toxic amitriptyline in the bloodstream. Oligochitosan derivatives with electron deficient aromatic rings were prepared with the reaction of benzenesulfonyl and dinitrobenzenesulfonyl groups under homogeneous conditions and characterized by 1H NMR, FT-IR, and MALDI-TOF. Blood-compatibility of functionalized oligochitosan was evaluated using thromboelastography. 1H NMR was also employed to elucidate the aromatic-aromatic interaction between electron deficient aromatic rings on oligochitosan and electron rich aromatic rings in amitriptyline. Experimental Section Materials. Oligochitosan (prepared by enzymatic degradation) was obtained from E-ZE Co. LTD (Korea) and used without further purification. The average molecular weight and moisture, as determined by the supplier, were 1150 Da and 8.0%, respectively. Dinitrobenzenesulfonyl chloride, benzenesulfonyl chloride, and amitriptyline were purchased from Aldrich and used as received. Chemical Modification. Before the reaction, the oligochitosan was dried in a vacuum. 3 g of oligochitosan powder were dissolved in dimethyl sulfoxide (15 mL) containing dinitrobenzenesulfonyl chloride (2.5 g) or benzenesulfonyl chloride (3.3 g). The reaction mixture was stirred and left for 2 days at room temperature. The resulting modified oligochitosan product was precipitated by adding ethanol and subsequently separated by centrifugation. The final product was Soxhlet-extracted with methanol overnight and dried in a vacuum. Characterization. 1H NMR spectra were obtained with a Varian VXR 300 (300 MHz) using D2O as a solvent. Chemical shifts were referenced to the internal water signal at 4.67 ppm. Finely ground oligochitosan derivatives were analyzed by using FT-IR (Nicolet Magna, U.S.A.) with a
KBr drift method. For MALDI-TOF analysis, oligochitosan was dissolved in H2O to which dihydroxylbenzoic acid was added as a matrix. The ratio of matrix to analyte is 100:1. 1 H NMR experiments for monitoring aromatic-aromatic interactions were carried out in D2O at 20, 37, and 50 °C. Stock solutions of oligochitosan and amitriptyline were prepared in D2O at the concentrations of 10 and 40 mM, respectively. Two stock solutions were mixed to give various ratios of oligochitosan to amitriptyline with a fixed concentration of oligochitosan, 5 mM. The ratio of oligochitosan to amitriptyline was varied from 1:1 to 1:4. Thromboelastography. Whole blood was obtained from 3 healthy male adults (22-31 year old). Thromboelastography (TEG) was carried out using a Thromboelastograph Coagulation Analyzer (model 3000C, Haemoscope, Co. Skokie, Illinois). Each oligochitosan (1.0 wt %) in a saline solution (30 µL) was pipetted into TEG cuvettes prewarmed to 37 °C. A CaCl2 solution of 30 µL and whole blood of 240 µL were added into the TEG cuvettes. The same volume of saline solution was used as a control. The pins of TEG were partially lowered and raised three or four times in order to ensure uniform mixing of the blood with the samples and standardize the agitation/activation of all blood specimens. Mineral oil was placed on the surface of the specimen to prevent air from drying out the samples. The TEG profile was obtained on a chart running at 2 mm/min. Results and Discussion Characterization. Benzenesulfonyl and dinitrobenzenesulfonyl groups were chosen as electron deficient aromatic rings. The chemical modification of oligochitosan was conducted under homogeneous conditions in DMSO. Figure 1 illustrates the chemical structure of oligochitosan and its derivatives. The resulting oligochitosan remained highly soluble in neutral water after the chemical modification. 1H NMR spectra of oligochitosan derivatives are shown in Figure 2. The signal at 1.7 corresponds to the acetyl protons of N-acetylglucosamine units.16 The protons at carbons (C-2) bearing amino groups are assigned to the signal at 2.8 ppm.24 The degree of deacetylation of the oligochitosan was calculated to be 65% from the integral intensity between
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Figure 3. FT-IR spectra of oligochitosan derivatives.
Figure 2. 1H NMR spectra of benzenesulfonyl (top) and dinitrobenzenesulfonyl (bottom) oligochitosan.
protons on C-2 and acetyl protons of N-acetyl-glucosamine. Further detailed assignment of all resonance signals was not attempted, because this would not add any great deal to this discussion. However, one important feature of these spectra is the appearance of new signals above 7 ppm. This region is typical for aromatic proton resonance. Benzenesulfonyl oligochitosan shows two signals with a clear distinction (gap) at 7.4 and 7.6 ppm, corresponding to meta and para protons and ortho protons, respectively. ortho protons are more deshielded than meta and para protons because of the magnetic anisotropy of the π bonds in the aromatic ring.25 The ratio of integral intensity of meta and para protons to ortho protons is 1.5:1. Dinitrobenzenesulfonyl oligochitosan shows three signals above 8 ppm with the identical integral intensity. The degree of substitution of oligochitosan was calculated to be 40% from the integral ratio between aromatic protons and protons at carbons (C-2). FT-IR spectra of oligochitosan derivatives are illustrated in Figure 3. The strong bands at approximately 1550 and 1600 cm-1 are attributed to the amide groups.26 The very strong band at 1550 cm-1 of dinitrobenzenesulfonyl oligochitosan is attributed to the asymmetric stretch of aromatic nitro groups. The asymmetric stretch of SdO in sulfonamide groups is represented at 1325 cm-1. The spectroscopic results demonstrate benzenesulfonyl and dinitrobenzenesulfonyl groups were successfully grafted to the oligochitosan backbone. The molecular weight of oligochitosan derivatives was measured by MALDI-TOF (Figure 4). MALDI-TOF has been used to investigate the biological molecules such as protein and enzyme and analyze the end-groups and repeating units of polymers up to 30 000 Da.27 Unmodified oligochitosan shows a broad molecular weight distribution (d.p. ) 3∼7). However, it is obvious that the difference of the
Figure 4. MALDI-TOF spectra of pure oligochitosan (top) and benzenesulfonyl oligochitosan (bottom).
adjacent two major peaks is 161. This corresponds to the molecular weight of one D-glucosamine unit, although chitosan consists of N-acetyl-D-glucosamine and D-glucosamine.28 It seems likely that benzenesulfonyl oligochitosan shows a similar pattern of molecular weight distribution. However, not only the molecular weight but also the discrepancy between adjacent two major peaks increased after the chemical modification due to the attachment of functional groups to oligochitosan molecules. It can also be speculated that oligochitosan was not depolymerized during the homogeneous chemical reaction because the portion of lower molecular weight did not increase after the chemical modification. Thromboelastography. Thromboelastography (TEG) measures the viscoelastic properties of blood as it is induced to clot under a low shear environment similar to sluggish blood flow in the body. TEG characterizes the formation and strength of the blood clot as a function of time.29 Formation of a clot is graphically represented as a characteristic cigar shape profile over time. TEG tracing profiles have some
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Figure 5. TEG tracings of bloods with saline solution (top) and dinitrobenzenesulfonyl oligochitosan.
principle parameters to be considered. Reaction time, R, is the period of time from initiation of the test to the initial fibrin formation. K is a measure of time from beginning of clot formation until the amplitude of thromboelastogram reaches 20 mm and represents the dynamics of clot formation. An R angle is an angle between the line in the middle of the TEG tracing and the line tangential to the TEG curve. Maximum amplitude (MA) is the maximum amplitude of the tracing and represents the strength of a clot.30,31 Figure 5 represents TEG profiles of blood with saline solution and dinitrobenzenesulfonyl oligochitosan solution. Saline solution was used as a negative control. No significant change was observed in the apparent shape, illustrating that unmodified benzenesulfonyl and dinitrobenzenesulfonyl oligochitosan are compatible with blood. TEG data from different functional groups were compared by the “Two Way Repeated Measures ANOVA”. A p value less than 0.05 was considered significant. There was no significant difference between oligochitosan-treated groups and saline solution-treated control. In addition, no significant difference was observed between unmodified oligochitosan and modified oligochitosan at the concentration of 1.0 and 0.1% in a saline solution. It should be noted that a preliminary experiment showed that benzenesulfonyl and dinitrobenzenesulfonyl oligochitosan did not manifest acute cardiotoxicity and weight gain or loss for 4 weeks, when intravenously administrated into rats via a tail vein. Aromatic-Aromatic Interaction. Amitriptyline has two aromatic rings with a high π-electron density because of the neighboring π electrons in the double bond and electron donating nature of methylene groups. Benzenesulfonyl and dinitrobenzenesulfonyl groups (Figure 1) have less π electron density compared to unsubstituted benzene rings because the sulfone and nitro groups are electron withdrawing. These electron rich and electron poor aromatic rings form com-
Figure 6. Complexation-induced variation in chemical shift of aromatic protons on oligochitosan derivatives.
plexes known as π-π complexes or donor-acceptor complexes.32 Various experimental methods have been adapted to examine π-π complexes. These include the vapor pressure osmometry, conductance, infrared spectroscopy, UV-vis absorbance, and nuclear magnetic resonance.33 Among these techniques, 1H NMR has been successfully used to examine the aromatic-aromatic interaction, because it is extremely sensitive to small changes in the electronic environment of a magnetic nucleus. In addition, it is experimentally easier to measure a peak position than an intensity in the spectrum.34 In 1H NMR, π-π complexation through the aromaticaromatic interaction is observed by monitoring the chemical shift of a given aromatic proton. Generally, chemical shift of protons on electron deficient aromatic rings (acceptors) is monitored when electron rich aromatic rings (donors) are present in a large excess.34 In the present work, oligochitosan concentration is 5 mM and the concentration of amitriptyline varies from 5 to 20 mM. The signal corresponding to meta and para protons of benzenesulfonyl groups was utilized to monitor the aromatic-aromatic interactions. Benzenesulfonyl aromatic protons were shifted upfield upon the addition of amitriptyline. The variation in chemical shift of aromatic protons in benzenesulfonyl oligochitosan is presented in Figure 6. Upfield shift of electron deficient aromatic protons can be explained by the magnetic anisotropy associated with an increased ring current shielding of electron rich aromatic
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rings.35 When electron deficient benzenesulfonyl rings form complexes with electron rich amitriptyline aromatic rings in a favored complexation geometry, benzenesulfonyl protons are more shielded by increased number of π electrons. Obvious increase in the chemical shift variation is observed with increasing amitriptyline concentration, suggesting that as the concentration of electron rich aromatic rings increases the more benzenesulfonyl groups participate in the π-π complexation. Ortho protons have a similar manner in chemical shift and the same magnitude of chemical shift variation. The largest variation observed by us is relatively low, 0.04 ppm (12 Hz), compared to the previously reported intermolecular π-π interaction.36,37 Some solute-solute complexes (mixed association) and self-association by π-π interactions or van der Waals force were reported to have a variation in chemical shift ranging from 25 to 250 Hz for 1 H NMR.33 It was suggested that the small change in the observed chemical shift is attributed to the high mobility of the counterpart aromatic rings with a slight preference for the complexation geometry.34 It appears that benzenesulfonyl rings have the temperature-dependent aromatic-aromatic interaction. Notable is that the largest variation of chemical shift is observed at the lowest temperature, 20 °C. This result can be rationalized by the fact that dissociation takes place at the high temperature because of the increased molecular kinetic energy which is unfavorable for association. It also seems likely that this aromatic-aromatic interaction is not strong enough to outweigh the reduced entropy caused by association at 37 and 50 °C.36 Dinitrobenzenesulfonyl oligochitosan shows a similar upfield behavior in the chemical shift change. The increased concentration of electron rich aromatic rings led to the increased chemical shift variation. All three different protons have the identical magnitude of chemical shift variation. However, dinitrobenzenesulfonyl protons have a larger variation than benzenesulfonyl protons at the same concentration of amitriptyline. This effect is due primarily to the strong electron-withdrawing nitro groups which result in the stronger aromatic-aromatic interaction. The presence of electron-withdrawing nitro groups decreases the π electron density over the aromatic ring, consequently, enhancing complexation through π electron donor-acceptor interactions. It has been also suggested that the closeness between aromatic rings is enhanced by the charge-induced dipole interactions.35 Based on this rationale, dinitrobenzenesulfonyl groups are supposed to form π-π complexes with a shorter distance between aromatic rings. In contrast to benzenesulfonyl oligochitosan, dinitrobenzenesulfonyl oligochitosan shows no temperature dependence in the aromatic-aromatic interaction. The magnitude of chemical shift variation was not influenced by temperature. To examine the dissociation of this π-π complex, the temperature was increased up to 80 °C. However, no temperature effect was observed in the variation of chemical shift. A reasonable explanation for this result involves consideration of association strength and molecular kinetic energy. As mentioned earlier, dinitrobenzenesulfonyl rings form strong π-π complexes through the complementary electrostatic interaction between electron deficient and electron rich aromatic rings. The magnitude
Lee and Baney
Figure 7. Proposed geometry of π-complexes of benzenesulfonyl (top) and dinitrobenzenesulfonyl (bottom) oligochitosans with amitriptyline.
of the complementary electrostatic interaction may be large enough to overcome the increased molecular kinetic energy which can cause them dissociate at high temperature.35 Aromatic-aromatic interactions have several different conformations (geometry) because the interactions result from different factors including electrostatic, hydrophobic, and van der Waals interaction.32,38 Benzene rings and substituted aromatic regions have been reported to form an aromatic stacking complex which is held together by electrostatic interactions of the π electron system.38,39 This suggests the structures in Figure 7 as one of possible models for π-π stacking of benzenesulfonyl and dinitrobenzenesulfonyl oligochitosan with amitriptyline. Conclusions Intravenously injectable oligochitosan derivatives bearing electron deficient aromatic rings were prepared for selective adsorption of tricyclic hydrophobic amitriptyline. Oligochitosan was chemically modified with benzenesulfonyl and dinitrobenzenesulfonyl rings under homogeneous conditions
Oligochitosan Derivatives
in dimethyl sulfoxide (DMSO). FT-IR and 1H NMR spectroscopy revealed that electron deficient aromatic rings were successfully grafted to the oligochitosan backbone. The degree of substitution was found to be 40% by 1H NMR by comparing the integral intensity of aromatic protons and protons on carbons (C-2) bearing amino groups. π-π complexation of electron rich amitriptyline aromatic rings with benzenesulfonyl or dinitrobenzenesulfonyl rings was examined by monitoring the chemical shift of aromatic protons. Aromatic protons in benzenesulfonyl and dinitrobenzenesulfonyl rings were upfield shifted with the addition of amitriptyline, supporting the π-π complexation through the aromatic-aromatic interaction. Dinitrobenzenesulfonyl rings have a smaller π electron density due to the presence of two electron-withdrawing nitro groups and form a stronger π-π complex through the electrostatic complementarity, thus, giving the larger variation in the chemical shift. Blood compatibility was accessed by thromboelastography (TEG). Both pure and modified oligochitosans did not affect the blood clotting. Acknowledgment. The authors acknowledge the financial support of the Particle Engineering Research Center (PERC) at the University of Florida, The National Science Foundation (NSF Grant EEC-94-02989), and the Industrial Partners of the PERC for support of this research. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect those of the National Science Foundation. References and Notes (1) Berthod, A.; Cremer, K.; Kreuter, J. J. Controlled Release 1996, 39, 17. (2) MacLaughlin, F. C.; Mumper, R. J.; Wang, J.; Tagliaferri, J. M.; Gill, I.; Hinchcliffe, M.; Rolland, A. P. J. Controlled Release 1998, 56, 259. (3) Shu, X. Z.; and Zhu, K. J. Int. J. Pharm. 2000, 201, 51. (4) van der Lubben, I. M.; Verhoef, J. C.; van Aelst, A. C.; Borchard, G.; Junginger, H. E. Biomaterials 2001, 22, 687. (5) Janes, K. A.; Fresneau, M. O.; Marazuela, A.; Fabra, A.; Alonso, M. J. J. Controlled Release 2001, 73, 255. (6) Hu, Y.; Jiang, X.; Ding, Y.; Ge, H.; Yuan, Y.; Yang, C. Biomaterials 2002, 23, 3193. (7) Remunan-Lopez, C.; Portero, A.; Lemos, M.; Vila-Jato, L.; Nunez, M. J.; Riveiro, P.; Lopez, J. M.; Piso, M.; Alonso, M. J. S. T. P. Pharm. Sci. 2000, 10, 69. (8) Calvo, P.; Remunan-Lopez, C.; Vila-Jato, J. L.; Alonso, M. J. J. Appl. Polym. Sci. 1997, 63, 125.
Biomacromolecules, Vol. 5, No. 4, 2004 1315 (9) Fernandez-Urrusuno, R.; Romani, D.; Calvo, P.; Vila-Jato, J. L.; Alonso, M. J. S. T. P. Pharma. Sci. 1999, 9, 429. (10) Kurita, K. Prog. Polym. Sci. 2001, 26, 1921. (11) Choi, W. S.; Ahn, K. J.; Lee, D. W.; Byun, M. W.; Park, H. J. Polym. Degrad. Stab. 2002, 78, 533. (12) Tokoro, A.; Tatawaki, N.; Suzuki, K.; Mikami, T.; Suzuki, S.; Suzuki. M. Chem. Pharm. Bull. 1988, 36, 784. (13) Richardson, S. C. W.; Kolbe, H. V. J.; Duncan, R. Int. J. Pharm. 1999, 178, 231. (14) Stone, C. K.; Kraemer, C. M.; Carroll, R. C.; Low, R. Ann. Emerg. Med. 1995, 26, 58. (15) Pozzo, A. D.; Fagnoni, L. V.; Guerrini, M.; Benedittis, D.; Muzzarelli, R. A. A. Carbohydr. Polym. 2000, 42, 201. (16) Kubota, N.; Tatsumoto, N.; Sano, T.; Toya, K. Carbohydr. Res. 2000, 324, 268. (17) Li, F.; Liu, W. G.; Yao, K. D. Biomaterials 2002, 23, 343. (18) Amiji, M. M. Carbohydr. Polym. 1997, 32, 193. (19) Shantha, K. L.; Harding, D. R. K. Carbohydr. Polym. 2002, 48, 247. (20) Murata, J.; Ohya, Y.; Ouchi, T. Carbohydr. Polym. 1996, 29, 69. (21) Sinnokrot M, O.; Valeev, E. F.; Sherrill, C. D. J. Am. Chem. Soc. 2002, 124, 10887. (22) Adams, H.; Blanco, J.-L. J.; Chessari, G.; Hunter, C. A.; Low, C. M. R.; Sanderson, J. M.; Vinter, J. G. Chem. Eur. 2001, 7, 3494. (23) Hunter, C. A. Chem. Soc. ReV. 1994, 23, 101. (24) Rinaudo, M.; Desbrieres, J.; Dung, P. L.; Binh, T.; Dong, N. T. Carbohydr. Polym. 2001, 46, 339. (25) Pavia, D. L.; Lampman, G. M.; Kriz, G. S. Introduction to spectroscopy, 2nd ed.; Sanders College Publishing: Fort Worth, TX, 1996. (26) Sugimoto, M.;, Morimoto, M.; Sashiwa, H.; Saimoto, H.; Shigemasa, Y. Carbohydr. Polym. 1998, 36, 49. (27) Brown, R. S.; Carr, B. L.; Lennon, J. J. J. Am. Soc. Mass Spectrosc. 1996, 7, 225. (28) Nah, J. W.; Jang, M. K. J. Polym. Sci. Part A: Polym. Chem. 2002, 40, 3796. (29) Mallet, S. V.; Cox, D. J. A. Thromboelastography; A Review Article. Br. J. Anaes. 1992, 69, 307. (30) Wenker, O. C.; Wojciechowski, Z.; Sheinbaum, R.; Zisman, E. Thromboelastography. Internet J. Anesth. 2000, 1. (31) Tobias, M. D.; Henry, C.; Augostides, Y. G. T. Lidocaine and bupivacaine exert differential effects on whole blood coagulation J. Clin. Anesth. 1999, 11, 52. (32) Hunter, C. A.; Sanders, J. K. M. J. Am. Chem. Soc. 1990, 112, 5525. (33) Ngowe, C. O.; Bishop. K. D.; McGuffin, V. L. Anal. Chim. Acta 2001, 427, 137-142. (34) Hanna, M. V.; Ashhaugh, A. L. J. Phys. Chem. 1964, 68, 811. (35) Jung, D. M.; de Ropp, J. S.; Ebeler, S. E. J. Agric. Food Chem. 2000, 48, 407. (36) Zych, A. J.; Iverson, B. L. J. Am. Chem. Soc. 2000, 122, 8898. (37) Funasaki, N.; Nomura, M.; Ishikawa, S.; Neya, S. J. Phys. Chem. 2001, 105, 7361. (38) Rashkin, M. L.; Waters, M. L. J. Am. Chem. Soc. 2002, 124, 1860. (39) Cubberley, M. S.; Iverson, B. L. J. Am. Chem. Soc. 2001, 123, 7560.
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