Bioconjugate Chem. 1994, 5,84-87
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Design and Synthesis of a Protein Device That Releases Insulin in Response to Glucose Concentration Yoshihiro Ito,' Dong-June Chung, and Yukio Imanishi Department of Polymer Chemistry, Faculty of Engineering, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto, Japan 606-01. Received June 3, 1993"
To synthesize a glucose-sensitive insulin-releasing protein device, insulin was esterified with methanol and connected to glucose oxidase with intervention of a disulfide compound, 5,5'-dithiobis(2-nitrobenzoic acid). On adding glucose to an aqueous solution containing the hybrid enzyme, the modified insulin was released. The amount of insulin released increased with increasing concentration of added glucose. The insulin release from the hybrid enzyme was specific to glucose. The activity of released insulin was about 80% of the unmodified insulin.
INTRODUCTION The control of drug release in response to external signals, e.g., pH change, temperature change, solubility change, antigedantibody reaction, or electric potential, has been investigated (1-4). We have been interested in the synthesis of an insulin-releasing device in response to glucose concentration as one of the drug-delivery systems that are responsive to physiological substances. Toward this goal, we have synthesized a membrane device to which insulin was connected with an intervening disulfide linkage together with glucose dehydrogenase (GDH),' nicotinamide adenine dinucleotide (NAD), and flavin adenine dinucleotide (FAD) ( 5 ) . In the presence of glucose, electrons generated in the oxidation reaction of glucose catalyzed by GDH are transferred to the disulfide linkage via immobilized NAD and FAD and undergo a reductive cleavage of the disulfide bond to release insulin. In the present investigation, the improvement of the efficiency of insulin release from the membrane device was aimed at, and a protein device was synthesized, in which insulin was connected to an enzyme, glucose oxidase (GOD), with an intervening disulfide linkage as shown in Figure 1. In contrast to GDH, GOD possesses a coenzyme FAD and functions without demanding the addition of FAD. Therefore, a more efficient use of electrons in the reductive cleavage of a disulfide linkage in the protein device than in the membrane device was expected. MATERIALS AND METHODS Reagents. The enzyme GOD (EC 1.1.3.4) from Aspergillus niger (G-8135,type X, 128IU/mg protein), insulin from bovine pancreas (1-5500, 24.4 IU/mg protein), and albumin of bovine origin (A-6003) were purchased from Sigma Chemical Co. (St. Louis, MO). 5,5'-Dithiobis(2nitrobenzoic acid) (DTNB), l-ethyl-3-(3-(dimethylamino)propy1)carbodiimide hydrochloride, which is a watersoluble carbodiimide (WSC), P-D(+)-glucose, galactose, maltose, urea, acrylamide, and a toluene solution of liquid scintillation were purchased from Nacalai Tesque Inc. ~~~
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Abstract published in Advance ACS Abstracts, December
15, 1993. Abbreviations: GDH, glucose dehydrogenase; NAD, nicotinamideadeninedinucleotide;FAD,flavin adeninedinucleotide; GOD, glucose oxidase; DTNB, 5,5'-dithiobis(2-nitrobenzoicacid); WSC, water-soluble carbodiimide; PBS, phosphate-buffered saline; GPC, gel permeation chromatography. 7 043-7 002/94/2905-0084$04.5Q/O
Figure 1. Insulin-releasingmechanism of the protein device in response to glucose addition, Ins representing insulin.
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NHCO.MNS-OOH
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W Figure 2. Synthetic scheme of the protein device. (Kyoto, Japan). 14C-Glucose (1-5 mCi/mmol) was purchased from DuPont (Wilmington, DE) and used for the measurement of biological activity of insulin. Phosphate-buffered saline (pH 7.0) was prepared by dissolving NaCl (8 g), KC1 (0.2 g), KHzP04 (0.2 g), NazHPO4 (1.15 g), MgC12 (0.1 g), and CaClz (0.1 g) into 1 L of distilled water. Synthesis of Insulin/GOD Hybrid. The insulin/ GOD hybrid was synthesized according to the scheme shown in Figure 2. First, in order to prevent inter- and intramolecular cross-linking reactions of proteins in the 0 1994 American Chemical Society
Qlucose-Sensitive Insulin-Releasing Proton Device WSC-activated reaction between insulin and DTNB or between DTNB-insulin and GOD, the carboxyl groups of insulin (500 mg) were esterified in 10% HC1-methanol (40 mL) a t room temperature for 24 h (6). The insulin methyl ester was precipitated from the reaction solution by adding anhydrous ether. The precipitate was dissolved in PBS and purified by using a gel permeation chromatography (GPC) column packed with Sephadex G-15 (eluent, PBS). The elution pattern was monitored with an absorbance a t 280 nm. The fractions of product, insulin methyl ester, were collected, desalted by dialysis, and finally freeze-dried. The product was a white powder (the yield was 99 % ) . The amount of carboxyl groups esterified with methanol was determined using 9-anthryldiazomethane (Funakoshi Co. Ltd., Tokyo). The dye (1mg) was dissolved in 50 pL of acetone and diluted with 450 pL of methanol. The dye solution was added into a sample solution, and the mixture was allowed to stand a t room temperature for 1h. After the sample was purified with a G-10 GPC column, the amount of dye coupled to the sample was measured by fluorescence. The relative emission intensity a t 412 nm was recorded when the excitation wavelength was 365 nm. DTNB (1g) was dissolved in water (50 mL) containing NazHP04 (0.827 g), and the solution was mixed with WSC (1g). To the resulting solution was added insulin methyl ester (150 mg) dissolved in 50 mL of water, and the mixture was stirred at 4 OC for 20 h. The reaction product was precipitated in methanol and purified by the same method using the G-15 GPC column (eluent, PBS). After the product was desalted and freeze-dried, a white powder was obtained (the yield was 69 % 1. The insulin methyl ester modified with DTNB (DTNBinsulin) (20 mg), WSC (34.8 mg), GOD (16.15 mg), and urea (2 M) was dissolved in 10 mL (pH 5.5) of 0.1 M boratebuffered solution, and the resulting solution was stirred a t 4 "C for 20 h. The presence of urea was based on the fact that ferrocene derivatives were efficiently coupled into the GOD to be electron mediators in the presence of urea as reported by Degani and Heller (7).After the urea was removed by ultrafiltration (Milipore membrane), the reaction product was purified by a G-50 GPC column (eluent, PBS) to collect the insulin/GOD hybrid. After the product was desalted and freeze-dried, a white powder was obtained (the yield was 61 % 1. Release of Insulin from the Insulin/GOD Hybrid. An aqueous sugar (glucose, galactose, or maltose) solution was added to a PBS (5 mL) containing the insulin/GOD hybrid (10 mg) at 37 OC. A portion of the solution was taken out after 1min, and the time-dependent amount of released insulin was determined on the basis of a 276-nm absorption intensity of the solution eluted through a reversed-phase column packed with a Biofine RPC-PO (JASCO, Tokyo, Japan). The determination was done with a calibration curve which was made by using known amounts of insulin. Biological Activity of Released Insulin. The biological activity of insulin was assayed by the determination of the amount of glucose taken up by adipocytes (8).An albumin-containing Krebs-Ringer bicarbonate buffer solution (1 mL) was added to adipocytes (mouse origin), and the mixture was incubated for 2 h in the presence of glucose/14C-glucose (100/1 mol/mol, total concentration, 8.3 pM, 100 pL) and an insulin derivative (12.5 mU/mL, 20 pL). The culture was terminated by adding 8 N H2SO4 (200 pL). Fat containing 14C-glucose,which was taken up by the cells, was extracted with a toluene liquid scintillation solution. The amount of 14Cwas determined by using an
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Figure 3. HPLC diagrams of unmodified GOD (A), untreated insulin (B),insulin/GOD hybrid (C), and insulin/GOD hybrid glucose (D):eluent, PBS (pH 7.0); column, Biofine RPC-PO (JASCO, Tokyo, Japan); flow rate, 0.5 mL/min; operating pressure, 30 kgf/cm2;detection, UV absorption at 276 nm.
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Aloka LSC 1000 scintillator (Tokyo, Japan) and compared with that in the incubation with native insulin to assess the relative biological activity of the insulin derivative. RESULTS AND DISCUSSION
Synthesis of the Insulin/GOD Hybrid. By the esterification reaction 55 % of the carboxylgroups in insulin were esterified with methanol. This was less than the value reported previously (6). When the DTNB was coupled to insulin, the elemental analysis of the reaction product of insulin and DTNB in a molar ratio of 1/5 gave the S content of 3.36%, indicating that two of the three amino groups in a molecule of insulin were used for the reaction with DTNB. When the DTNB-insulin was coupled to GOD, the elemental analysis of the reaction product of DTNBinsulin and GOD in a molar ratio of 1/1 showed the S content of 2.27 % ,indicating that 20 mol % of GOD amino groups was used for coupling with DTNB-insulin and 5.8 insulin molecules were included in the hybrid. On the other hand, by cleavingwith 0.5 vol 5% of mercaptoethanol 6.3 insulin molecules were released. The molecular weight of the hybrid was estimated to be 193 kDa by GPC (Cosmosil/Cosmogelmade by Nacalai Tesque, Inc., Kyoto, Japan). This result indicates that 6.6 insulin molecules were linked to a GOD molecule. These results demonstrated that about six insulin molecules were linked to GOD through each disulfide bond of DTNB. Insulin Release upon Glucose Addition. Highperformance liquid chromatography diagrams of aqueous solutions containing the insulin/GOD hybrid or unmodified insulin before and after glucose addition are shown in Figure 3. A new peak appeared on adding glucose in the solution containing the insulin/GOD hybrid. The retention time of the new peak almost agreed with that
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Table 1. Biological Activities of Insulin Derivatives re1 biological re1 biological insulin deriv activity’ (%) insulin deriv activity’ (%) native insulin 100 16 DTNB-Ins 73 20 Me-Ins 95 9 released insulin 81 f 9
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a The relative biological activity is normalized by the activity of native insulin as a standard, 100%.
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Figure 4. Time-dependent insulin release from the insulin/ GOD hybrid in response to glucose addition (15 mM).
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Figure 5. Release of insulin from the insulin/GOD hybrid in response to different concentrations of glucose added (0.15,1.5, 15, 30,60,and 150 mM). for the unmodified insulin, although the new peak was not completely the same as that of the native insulin since the released insulin was methyl esterified and carried 2-nitro-5-mercaptobenzonic acid. The retention times of both A and B chains of insulin, which were obtained by the degradation method reported previously (91, were markedly longer than that of the native insulin. These observations imply that the insulin/GOD hybrid released insulin upon addition of glucose without side reactions, e.g., a cleavage of disulfide linkages of insulin itself. This conclusion was also deduced from the biological activity described below. The time-dependent release of modified insulin from the insulin/GOD hybrid was measured, and the result is shown in Figure 4. The insulin was immediately released from the hybrid on adding an aqueous glucose solution. The amount of insulin released 30 min after the addition of glucose solution of varying concentrations was measured, and the data are summarized in Figure 5. The amount of released insulin increased with increasing concentration of glucose solution. However, the rate of insulin release was not controlled by the concentration of glucose added. It was considered that the insulin would have a maximal rate as long as the glucose concentration was higher than its K, with glucose oxidase. The number of modified insulin molecules released on oxidation of a glucose molecule was calculated to be 1.3
which x 10-3. This value is nearly 40-fold of 3.4 X was the number of insulin molecules released per a molecule of glucose oxidized in the previously reported membrane device (5). The comparison shows that the protein device is more efficient in releasing insulin than the membrane device. Sugars other than glucose did not induce insulin release from the insulin/GOD hybrid. This result indicates a glucose specificity of the insulin/GOD hybrid. In addtion to the experiment with 2-mercaptoethanol, in order to confirm that the modified insulin was released after reductive cleavage of disulfide bond, NADH and FADHz instead of glucose were added to the solution containing the protein device. The modified insulin was also released by the addition of reducing agents. These observations were also previously reported with the membrane system using glucose dehydrogenase (5). Biological Activity of Released Insulin. Biological activities of insulin methyl ester, DTNB-insulin, and insulin/GOD hybrid were determined by the 14C-glucose uptake method and are shown in Table 1 relative to that of unmodified insulin. The biological activity of insulin decreased slightly by the esterification with methanol and to 73 % by the connection of DTNB. The biologicalactivity of the released insulin was 81% of that of unmodified insulin. The relative biological activities are comparable to those of insulins modified with sugars (IO). The different activities between the released insulin and the DTNB-insulin could be explained in terms of different bulkiness of substituents connected to amino groups of insulin. This hybrid is a prototype of a protein device working like a molecular machine. For practical use, this protein device will be encapsulated by a semipermeable membrane through which the released insulin can permeate, or will be modified with polyethylene glycol, thus being immunoisolated from biocomponents such as immunoglobulin in the body. LITERATURE CITED (1) Ito, Y. (1992) Enzyme modification for drug delivery molecular devices. In Syntheis of Biocomposite Materials (Y. Imanishi, Ed.) pp 137-180, CRC Press, Boca Raton. (2) Ito,Y.,Casolaro,M.,Kono,K.,andImanishi,Y. (1989)Design and synthesis of insulin-releasing system in response to glucose. J . Controlled Release 10, 195-202. (3) Ito, Y., Kotera, S., Inaba, M., Kono, K., and Imanishi, Y. (1990) Control of poly(acry1ic acid) grafts of pore size of polycarbonate membrane with straight pores. Polymer 31,
2157-2161. (4) Ito, Y., Inaba, M., Chung, D. J., and Imanishi, Y. (1992) Control of water permeability by pH and ionic strengththrough a porous membrane having poly(carboxy1ic acid) surfacegrafted. Macromolecules 25, 7313-7316. (5) Chung, D. J.,Ito,Y., andImanishi, Y. (1992)Glucose-sensitive
insulin-releasing system using redox reaction. J. Controlled Release 18, 45-54. (6) Levy, D., and Carpenter, F. H. (1970) Insulin methyl ester. Specific cleavage of a peptide chain resulting from a nitrogen to oxygen acyl shift at a threonine residue. Biochemistry 9, 3215-3222.
Glucose-Sensitive Insulin-Releasing Proton Device
(7) Degani, Y. and Heller, A. (1988) Direct elecrical communi-
cation between chemically modified enzymes and metal electrodes. 2. Methods for bonding electron-transfer relays to glucose oxidase and D-amino-acidoxidase.J.Am. Chem. SOC. 110, 2615-2620. (8) Moody, A. J., Stan, M. A,, Stan,M., and Glieman, J. (1974) A simple free fat cell bioassay for insulin. Horm. Metab. Res. 6 , 12-16.
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(9) Florence,T. M. (1980) Degradationof protein disulfidebonds in dilute alkali. Biochem. J. 189, 507-520.
(10)Seminoff, L. A., Gleeson, J. M., Zheng, J., Olsen, G. B., Holmberg, D. L., Mohammad, S. F., Wilson, D., and Kim, S. W. (1989) A self-regulating insulin delivery system. 11. In vivo characteristics of a synthetic glycosylated insulin. Int. J. Pharm. 54, 251-257.
