超氧化物歧化酶明胶微球的制备与表面化学修饰研究
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摘要
超氧化物歧化酶(SOD)是重要的氧自由基清除剂，可以用于治疗和预防与自由基损伤有关的一些疾病。目前，SOD在临床应用上主要以注射方式给药，而口服给药方式研究较少。本工作将SOD制成微球，以提高SOD稳定性和口服相对生物利用度，且在微球制备条件方面作了进一步研究。
以连苯三酚自氧化法作为SOD的活力分析方法，并对该方法的精密度，回收率及线性范围进行了考察。结果表明，该方法可行。
从理化性质的角度，考察了不同高分子材料对SOD活力的影响，结果表明天然高分子材料更有助于保持SOD的活力。
本工作采用乳化凝聚法制备明胶微球。对制备条件先进行了单因素考察，然后利用正交设计进行了优选。最佳制备条件为：水相油相体积比为3：40，温度为60℃，时间为5min，转速为400rpm；所得微球载药量为23200u／g，包封率为88％，粒径分布集中在25～75um。
本工作考察了海藻酸钠的流变性，与明胶的相容性，以及与明胶的界面张力，并测定了壳聚糖的脱乙酰度和分子量。在此基础上制备了明胶海藻酸钠复合微粒及表面修饰的明胶微球，对它们的各种性质，包括含药量，稳定性，释放性及表面性质进行了研究。
在药效学方面，首先考察了SOD抗小鼠耳肿胀作用的量效关系，然后考察了SOD溶液和不同形式微球抗炎作用的时效关系。结果表明，几种微球均可显著延长SOD抗炎作用的时间。
Superoxide dismutase (SOD) is an important scanvenger of reactive oxygen radicals, which can be used to treat or prevent radical-related diseases. Nowadays, SOD is used mainly as injection clinically, while oral formulation for SOD is less investigated. In this work, SOD gelatin microspheres were made with the aim to improve its stability, oral relative bioavailability, and to study the preparation conditions of microspheres.
First, the auto-oxidization of pyrogallol was used as the analysis method to determine SOD activity, and the precision , recovery and linear range of the method were investigated. The results showed that the method was feasible.
In physical and chemical property tests, different macromolecular materials were tried to protect SOD activity, and the results showed that natural polymers, such as gelatin and alginate, were more effective in SOD activity protection than synthesized ones.
In this study, SOD gelatin microspheres were prepared by emultion coagulation procedure. After single factor tests, the orthogonal design were performed to opitimize the conditions for the process. The best condition was W/O,3: 40, temperature, 60 C, time, 5 min, agitation rate, 400rpm. The microspheres thus derived had an embedding rate of 88%, a drug loading of 23200u/g, and a size mainly around 25-75 urn.
We studied the rheology of alginate, its compatability with gelatin and the interfacial tension between alginate and gelatin, the molecular weight and degree of deacylation of chitosan were determined as well. On the basis of the work above,
the alginate gelatin complex microparticle and the surface modified microspheres
were prepared, and the SOD embedding rate ,SOD stability and release behavior of the product were determined . The surface property of the modified microsphere were studied, too.
In pharmacodynamics, the dose-response relationship of SOD in antiinflammation was studied, then the time-response property of different microspheres were investigated. The results showed that all of the microspheres could prolong the antiinflammation time in mouse.
以连苯三酚自氧化法作为SOD的活力分析方法，并对该方法的精密度，回收率及线性范围进行了考察。结果表明，该方法可行。
从理化性质的角度，考察了不同高分子材料对SOD活力的影响，结果表明天然高分子材料更有助于保持SOD的活力。
本工作采用乳化凝聚法制备明胶微球。对制备条件先进行了单因素考察，然后利用正交设计进行了优选。最佳制备条件为：水相油相体积比为3：40，温度为60℃，时间为5min，转速为400rpm；所得微球载药量为23200u／g，包封率为88％，粒径分布集中在25～75um。
本工作考察了海藻酸钠的流变性，与明胶的相容性，以及与明胶的界面张力，并测定了壳聚糖的脱乙酰度和分子量。在此基础上制备了明胶海藻酸钠复合微粒及表面修饰的明胶微球，对它们的各种性质，包括含药量，稳定性，释放性及表面性质进行了研究。
在药效学方面，首先考察了SOD抗小鼠耳肿胀作用的量效关系，然后考察了SOD溶液和不同形式微球抗炎作用的时效关系。结果表明，几种微球均可显著延长SOD抗炎作用的时间。
Superoxide dismutase (SOD) is an important scanvenger of reactive oxygen radicals, which can be used to treat or prevent radical-related diseases. Nowadays, SOD is used mainly as injection clinically, while oral formulation for SOD is less investigated. In this work, SOD gelatin microspheres were made with the aim to improve its stability, oral relative bioavailability, and to study the preparation conditions of microspheres.
First, the auto-oxidization of pyrogallol was used as the analysis method to determine SOD activity, and the precision , recovery and linear range of the method were investigated. The results showed that the method was feasible.
In physical and chemical property tests, different macromolecular materials were tried to protect SOD activity, and the results showed that natural polymers, such as gelatin and alginate, were more effective in SOD activity protection than synthesized ones.
In this study, SOD gelatin microspheres were prepared by emultion coagulation procedure. After single factor tests, the orthogonal design were performed to opitimize the conditions for the process. The best condition was W/O,3: 40, temperature, 60 C, time, 5 min, agitation rate, 400rpm. The microspheres thus derived had an embedding rate of 88%, a drug loading of 23200u/g, and a size mainly around 25-75 urn.
We studied the rheology of alginate, its compatability with gelatin and the interfacial tension between alginate and gelatin, the molecular weight and degree of deacylation of chitosan were determined as well. On the basis of the work above,
the alginate gelatin complex microparticle and the surface modified microspheres
were prepared, and the SOD embedding rate ,SOD stability and release behavior of the product were determined . The surface property of the modified microsphere were studied, too.
In pharmacodynamics, the dose-response relationship of SOD in antiinflammation was studied, then the time-response property of different microspheres were investigated. The results showed that all of the microspheres could prolong the antiinflammation time in mouse.
引文
[1] 王夔，许锦善，李荣昌，医化学基础——疾病发生发展的化学规律，北京，北京大学出版社，2001，91-106
[2] 袁勤生，SOD在医药，食品和日化工业上的应用，中国生化药物杂志，1994，15(4)，289-293
[3] Daneil R.Rosen, Teepu Siddique, David Patterson, etc., Mutations in Cu,Zn-SOD gene are associated with familial amyotrophic lateral sclerosis, Nature, 1993, 362, 59-62
[4] 徐涌铭节译，Orgotein，国外药学(合成药，生化药，制剂分册)，1982，3，52
[5] a．吴景时等，新药手册，第一版，中国科学技术出版社，北京，1998，130
b．孙定人，张石革，梁之江，国家临床新药集，中国医药科技出版社，北京，2001，758
[6] John A.Tainer, Elizabeth D.Getzoff, Karl M.Beem, etc., Determination and analysis of the 2(?) structure of copper, zinc superoxide dismutase, J. Mol. Biol., (1982) 160, 181-217
[7] http://srs.dl.ac.uk/arch/talks/hough/sod-strycture_seminar/sld036.htm
[8] 王凤山，凌沛学，生化药物研究，人民卫生出版社，北京，1997，459
[9] a. D. Nakae, H. Yoshiji, T. Amanuma, et al. Endocytosis-independent uptake of liposome-encapsulated superoxide dismutase prevents the killin of cultured hepatocytes by tert-butyl hydroperoxide. Arch. Biochem. Biophys. 279 (1990) 315-319.
b. C. Regnault, M. Roch-Arveiller, M. Tissot, et al. Effect of encapsulation in liposome on the anti-inflammatory properties of superoxide dismutase after oral administration, Clin. Chim. Acta 240 (1995) 117-127.
[10] a. T. Fujita, H.Furutsu, M.Nishikara, et al., Therapeutic effects of superoxide dismutase derivatives modified with mono-or polysaccharides on hepatic injury induced by ischemia/reperfusion,Biochem. Biophys.Res.Commun, 189 (1992) 191-196.
b. A.V. Maksimenko, L.M. Bezrukavnikova, E.L. Grigoryeva, et al., Effect of native and modified forms of superoxide dismutase and catalase on experimental silicosis in rats, Ann. N.Y. Acad. Sci.672 (1992) 118-125.
[11] a. P. Caliceti, O. Schiavon, M. Morpuro, et al., Physico-chemical and biological of monofunctional hydroxy terminating poly(N-vinyl pyrrolidone) conjugated superoxide dismutase, J. Bioact. Compat. Polym. 10 (1995) 103-105.
b.P.Caliceti, O. Schiavon, T. Hirano, et al., Modification of physico-chemical and biopharmaceutical properties of superoxide dismutase by conjugation to the copolymer of divinyi ether and maleic anhydride, J.Controlled Release 39 (1996) 27-34.
[12] a. C. Yamazaki, J. Hoshino, Y. Hori, et al., Effect of lecithinized-superoxide dismutase on the interstitial pneumonia model induced by blemycin in mice, Jpn. J. Pharmacol. 75 (1997) 97-100.
b. T. Nakamura, R. Igarashi, T. Kurashina, et al., Lecithinized superoxide dismutase iinduces vasodilation: evidenceof direct contribution of superoxide anions to modulating vascular tone, Life Sci. 64 (1999) 65-70.
c. T. Miyahara, T. Shibamoto, H.G. Wang, et al., lecithinized superoxide dismutase attenuates phorbol myristate acetate-induced injury in dog lung, Eur. J. Pharmacol. 344 (1998) 231-239.
d. M. Takenaga, R. lgarashi, A. Ochiai, et al., Effect of lecithinized superoxide dismutase (PC-SOD) on experimental pulmonary metastasis in mice, Free Radic. Biol. Med. 26 (1999) 1117-1125
[13] a. K. Mihara, Y. Oka, K. Sawai, et al. Improvement of therapeutic effect of human recombinant superoxide dismutase on ischaemic acute renal failure in the rat via cationization and conjugation with polyethylene glycol, J. Drug Target. 2 (1994) 317-321.
b. F.M. Veronese, P. Caliceti, A. Pastorino, et al. Preparation, physico-chemical and
pharmacokinetic characterization of monomethoxypoly(ethylene glycol)-derivatized superoxide dismutase, J. Controlled Release 10 (1989) 145-154.
c. A. Conforti, L. Sartore, et al. Anti-inflammatory activity of monomethoxypoly(ethylene glycol) superoxide dismutase on adjuvant arthritis in rats, Pharmacol. Res. 23 (1991) 51-56.
[14] a．谢英，周轩，王军波，等，铜-(N-正十二碳酰双甘肽)配合物模拟SOD活性的研究，高等学校化学学报，2001，22(1)，67-69
b．罗慧勤，铜锌超氧化物歧化酶的模拟化学研究，高等学校化学学报，1997，18(7)，1012-1018
c．谢英，金虬，李大珍，等，停流法研究Cu(Ser)_2及Cu(Gly-Gly)_2催化O~-歧化反应动力学，高等学校化学学报，1998，19(8)，1288-1291
[15] 黄维华，袁勤生，口服超氧化物歧化酶在生物体中的吸收中国生化药物杂志，1995，16(5)，201-204
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[22] 廖公铁，靶向给药制剂，四川科学技术出版社，成都，1997，81-111
[23] 徐希明，张均寿，微球给药系统的工艺优化研究，中国现代应用药学杂志，1996，16(3)，1-4
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on PLGA microspheres and PVA nanoparticles for long-term protein drug delivery, PharmaceuticaI Research, 1999, 16 (9), 1430-1435
b. Jia KuiLi, Muo Wang, Xue Shen Wu, A novel biodegradable system based on gelatin nanoparticle and PLGA micmspheres for protein and peptide drug delivery, J.Pharm.Sci.1997, 86 (8), 891-895
[26] Kathleen J. Pekarek, Jules S. Jacob, Edith Mathiowitz, Double-walled polymer microspheres for controlled drug release, Nature, 1994, 367, 258-260
[27] Shen-YangLin,Pei-ChinLin,Effect of the acid type,acetic acid and CMC-Na concentrations on the formation,micromeritic,dissolution and floating properties of theophylline chitosan microcapsules, Chem. Pharm. Bull.,40(9),2491-2497,1992
[28] 莫秀梅，甲壳胺-明胶共混物的相容性研究，高分子学报，1997，2，222-224
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[2] 袁勤生，SOD在医药，食品和日化工业上的应用，中国生化药物杂志，1994，15(4)，289-293
[3] Daneil R.Rosen, Teepu Siddique, David Patterson, etc., Mutations in Cu,Zn-SOD gene are associated with familial amyotrophic lateral sclerosis, Nature, 1993, 362, 59-62
[4] 徐涌铭节译，Orgotein，国外药学(合成药，生化药，制剂分册)，1982，3，52
[5] a．吴景时等，新药手册，第一版，中国科学技术出版社，北京，1998，130
b．孙定人，张石革，梁之江，国家临床新药集，中国医药科技出版社，北京，2001，758
[6] John A.Tainer, Elizabeth D.Getzoff, Karl M.Beem, etc., Determination and analysis of the 2(?) structure of copper, zinc superoxide dismutase, J. Mol. Biol., (1982) 160, 181-217
[7] http://srs.dl.ac.uk/arch/talks/hough/sod-strycture_seminar/sld036.htm
[8] 王凤山，凌沛学，生化药物研究，人民卫生出版社，北京，1997，459
[9] a. D. Nakae, H. Yoshiji, T. Amanuma, et al. Endocytosis-independent uptake of liposome-encapsulated superoxide dismutase prevents the killin of cultured hepatocytes by tert-butyl hydroperoxide. Arch. Biochem. Biophys. 279 (1990) 315-319.
b. C. Regnault, M. Roch-Arveiller, M. Tissot, et al. Effect of encapsulation in liposome on the anti-inflammatory properties of superoxide dismutase after oral administration, Clin. Chim. Acta 240 (1995) 117-127.
[10] a. T. Fujita, H.Furutsu, M.Nishikara, et al., Therapeutic effects of superoxide dismutase derivatives modified with mono-or polysaccharides on hepatic injury induced by ischemia/reperfusion,Biochem. Biophys.Res.Commun, 189 (1992) 191-196.
b. A.V. Maksimenko, L.M. Bezrukavnikova, E.L. Grigoryeva, et al., Effect of native and modified forms of superoxide dismutase and catalase on experimental silicosis in rats, Ann. N.Y. Acad. Sci.672 (1992) 118-125.
[11] a. P. Caliceti, O. Schiavon, M. Morpuro, et al., Physico-chemical and biological of monofunctional hydroxy terminating poly(N-vinyl pyrrolidone) conjugated superoxide dismutase, J. Bioact. Compat. Polym. 10 (1995) 103-105.
b.P.Caliceti, O. Schiavon, T. Hirano, et al., Modification of physico-chemical and biopharmaceutical properties of superoxide dismutase by conjugation to the copolymer of divinyi ether and maleic anhydride, J.Controlled Release 39 (1996) 27-34.
[12] a. C. Yamazaki, J. Hoshino, Y. Hori, et al., Effect of lecithinized-superoxide dismutase on the interstitial pneumonia model induced by blemycin in mice, Jpn. J. Pharmacol. 75 (1997) 97-100.
b. T. Nakamura, R. Igarashi, T. Kurashina, et al., Lecithinized superoxide dismutase iinduces vasodilation: evidenceof direct contribution of superoxide anions to modulating vascular tone, Life Sci. 64 (1999) 65-70.
c. T. Miyahara, T. Shibamoto, H.G. Wang, et al., lecithinized superoxide dismutase attenuates phorbol myristate acetate-induced injury in dog lung, Eur. J. Pharmacol. 344 (1998) 231-239.
d. M. Takenaga, R. lgarashi, A. Ochiai, et al., Effect of lecithinized superoxide dismutase (PC-SOD) on experimental pulmonary metastasis in mice, Free Radic. Biol. Med. 26 (1999) 1117-1125
[13] a. K. Mihara, Y. Oka, K. Sawai, et al. Improvement of therapeutic effect of human recombinant superoxide dismutase on ischaemic acute renal failure in the rat via cationization and conjugation with polyethylene glycol, J. Drug Target. 2 (1994) 317-321.
b. F.M. Veronese, P. Caliceti, A. Pastorino, et al. Preparation, physico-chemical and
pharmacokinetic characterization of monomethoxypoly(ethylene glycol)-derivatized superoxide dismutase, J. Controlled Release 10 (1989) 145-154.
c. A. Conforti, L. Sartore, et al. Anti-inflammatory activity of monomethoxypoly(ethylene glycol) superoxide dismutase on adjuvant arthritis in rats, Pharmacol. Res. 23 (1991) 51-56.
[14] a．谢英，周轩，王军波，等，铜-(N-正十二碳酰双甘肽)配合物模拟SOD活性的研究，高等学校化学学报，2001，22(1)，67-69
b．罗慧勤，铜锌超氧化物歧化酶的模拟化学研究，高等学校化学学报，1997，18(7)，1012-1018
c．谢英，金虬，李大珍，等，停流法研究Cu(Ser)_2及Cu(Gly-Gly)_2催化O~-歧化反应动力学，高等学校化学学报，1998，19(8)，1288-1291
[15] 黄维华，袁勤生，口服超氧化物歧化酶在生物体中的吸收中国生化药物杂志，1995，16(5)，201-204
[16] 平其能，现代药剂学，中国医药科技出版社，北京，1998，669
[17] Marklund S, Marklund G, Eur. J. Biochem, 1974, 47, 469
[18] 静天玉，赵晓瑜，用终止剂改进超氧化物歧化酶连苯三酚测活法，生物化学与生物物理学进展，1995，22(1)，84-87
[19] 陈浩，朱利民，袁勤生，Cu，Zn-SOD某些理化性质的研究，生化药物杂志，1991，1，27
[20] 胶体与界面化学实验，北京大学出版社，北京，1993，19，330，19
[21] 胶体与界面化学实验，北京大学出版社，北京，1993，19，330，300
[22] 廖公铁，靶向给药制剂，四川科学技术出版社，成都，1997，81-111
[23] 徐希明，张均寿，微球给药系统的工艺优化研究，中国现代应用药学杂志，1996，16(3)，1-4
[24] 高聚物的结构与性能，科学出版社，北京，1983，165
[25] a. Muo Wang, Xue Shen Wu, Jia KuiLi, A heterogeneously structured composite based
on PLGA microspheres and PVA nanoparticles for long-term protein drug delivery, PharmaceuticaI Research, 1999, 16 (9), 1430-1435
b. Jia KuiLi, Muo Wang, Xue Shen Wu, A novel biodegradable system based on gelatin nanoparticle and PLGA micmspheres for protein and peptide drug delivery, J.Pharm.Sci.1997, 86 (8), 891-895
[26] Kathleen J. Pekarek, Jules S. Jacob, Edith Mathiowitz, Double-walled polymer microspheres for controlled drug release, Nature, 1994, 367, 258-260
[27] Shen-YangLin,Pei-ChinLin,Effect of the acid type,acetic acid and CMC-Na concentrations on the formation,micromeritic,dissolution and floating properties of theophylline chitosan microcapsules, Chem. Pharm. Bull.,40(9),2491-2497,1992
[28] 莫秀梅，甲壳胺-明胶共混物的相容性研究，高分子学报，1997，2，222-224
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