碱性成纤维生长因子纳米给药系统的研究
摘要
生物多肽及蛋白质类药物因其独特的生物活性以及与化学药物相比更安全的特点,越来越得到广泛的关注和应用。由于该类药物的稳定性普遍较差,传统的药物剂型很难维持其在体内的稳定性,且临床上大多采用注射给药方式,长期频繁给药会给患者造成很大的痛苦,并增加经济负担。因此开发和研究多肽类药物的新型给药系统,以提高药物疗效、方便患者治疗,是药剂学研究中有待开发的新领域,具有明显的社会效应和经济价值。
冠心病是目前人类死亡的主要疾病之一,在我国其发病率呈逐年增高趋势,越来越多医学工作者在不断寻求新的冠心病治疗方法,而治疗性血管生成(therapeutic angiogenesis)或称“生物搭桥”(biologic bypass)的方法是目前冠心病治疗研究的热点。近年来,随着分子生物学的发展,人们发现血管新生过程中,有许多细胞因子的参与,而碱性成纤维生长因子(basic fibroblast growth factors, bFGF)就是其中之一。bFGF作为成纤维细胞生长因子(fibroblast growth factors, FGF)中的一种类型,发挥着它在各方面的功效。外源性bFGF能够促进细胞分裂、趋化,诱导胚胎正常发育、创面愈合、神经再生及功能恢复和促进血管生长。但是bFGF在体内的半衰期为3～10分钟,全身应用将被快速灭活而难以发挥其有效作用;在溶液中的稳定性较差,局部穿刺分次给药也难以产生有效作用,并且不适合长期给药。因此研究bFGF的纳米粒对长期维持局部药物的有效浓度,促进新生血管生长有着重要意义。
本研究以碱性成纤维生长因子(bFGF)为模型药物,系统地研究了bFGF纳米粒的制备处方和工艺,考察了制剂的粒径及分布、包封率及载药量和体外释药的特点,并对药物的体外活性进行了初步的研究。
利用酶联免疫吸附法(Enzyme Linked Immunosorbent Assay,ELISA)对碱性成纤维生长因子的含量进行测定,研究了碱性成纤维生长因子在制备条件下的稳定性。结果表明:药物在冷冻条件下保存最稳定,至少可保存一个月不降解;冷藏条件下保存,7天后药物浓度有所下降,药物活性部分丧失,故该药制成品宜冷冻保存。在4～15℃范围内、pH值在2.0～7.0之间以及搅拌速度为880r?min-1搅拌2h的条件下,制备成的纳米粒不会影响碱性成纤维生长因子的生物活性。
以粒径及其分布和包封率为指标,比较了三种载体材料制备工艺上的差异以及在质量上的区别,由实验结果可得出,以α-氰基丙烯酸正丁酯为载体的纳米粒包封率最高、制备方法简单、耗时最短,从而筛选出本研究所采用的载体材料α-氰基丙烯酸正丁酯。同时还得到得到粒径为89.9nm,包封率为28.13%的PLA-bFGF-NP和粒径为453nm,包封率为48%的CS-bFGF-NP。单因素实验考察了表面活性剂的种类和用量、载体材料的用量、药物与材料比例、pH值、搅拌速度等对碱性成纤维生长因子的影响,在单因素实验考察基础上,采用正交设计优化处方和制备工艺,得到粒径为120.5nm,包封率为89.35%的PBCA-bFGF-NP。
α-氰基丙烯酸正丁酯纳米粒的体外释放实验中,2h内释放小于30%,没有突释效应,而释放70%需要3天左右,具有明显的缓释效果,胶体溶液的释放可用双指数双相动力学方程或多项式方程描述,相关系数为分别为0.9905和0.9947。
在体外活性实验中,以鸡胚尿囊膜( Chorioallantoic Membrane , CAM )为动物模型,以无菌生理盐水作为阴性对照组,bFGF原料药为阳性对照组,分别比较高、中、低剂量PBCA-bFGF-NP对CAM血管增生的影响。实验结果显示:PBCA-bFGF-NP能够促进血管增生;统计结果表明:高、中剂量组与阴性对照组、低剂量组有明显的差异,而高、中剂量组和阳性对照组没有明显的差异。
本研究通过应用新型高分子材料和纳米粒制备技术,得到PBCA-bFGF-NP,其缓释效果达到项目设计要求。研究工作丰富了蛋白多肽类药物的缓释给药系统研究内容,具有较大的理论意义和广阔前景,具有较好的应用开发价值。
With the booming development and widely application of polypeptide and protein as an important method using in clinic, more and more urgent request are put through for a new drug delivery system of this kind of medicine instead of traditional pharmaceutical dosage form. To overcome the disadvantage of traditional form of medication, such as the pain has to suffer and the expensive fees in the curing process, a new drug new drug delivery system has to be found. It is necessary and full of esperance for both the medicine society and the pharmaceutical manufactures.
Nanoparticle has been regarded as a useful vector for targeted drug delivering system recent years. Many people pay attention to on the nanoparticles formed of biodegraded materials, such as polylactide (PLA), chitosan and butyl-2-cyanoacrylate (BCA), et al. We use them to protect drug form being degraded and delivery the drug into blood circle.
The basic fibroblast growth factors (bFGF) is a type of the fibroblast growth factors (FGF), it brought effect in many fieds. The ectogenesis basic fibroblast growth factors can promote the cell division and chemotaxis, and induce the normal growth of embryo, and promote the growth of blood vessel, create to regeneration of nerve and healing of wound. But the half-life of bFGF is about 3-5 minutes in bodys. If it is applied to whole body ,it is quickly caused to inactivation and hard to develope its valid function. The bFGF is unstability in solutions. It is very difficult to effect that it is applied many times though local puncture, and bFGF is not suitable for over a long period of time to the medicine. So it is very important significance that to study on sustained release bFGF nanoparticle of maintenancing the local effect of drug and promoting the the growth of blood vessel.
By using enzyme linked immunosorbent assay (ELISA) method , we studied the effect of different temperature、pH value and the rotation speed upon bFGF under the preparation condition. The optimum maintaining condition is to be preserved by freezing. No matter bathed in 37℃water for 24 hours or kept in tri-hydroxyl aminomethane buffer solution (Tris) (pH2.0) under 15℃for 1 hours, bFGF can not degraded. And the rotation speed of 880 revolutions per minute for 2 hours is safe for the preparation. The experiment indicates that bFGF is stable enough to survive through the whole preparation process.
The effects of different concentration of surfactant and polymer, the drug: polymer ratio and the pH value in the nanoparticle forming process were investigated. All these factors influence the size and entrapment efficiency obviously. Respectively, we use three kinds of method to optimize the formulation of the prescription and obtained PLA-bFGF-NP, which diameter is 89.9 nm and EE is28.13% , CS-bFGF-NP that diameter is 453nm and EE is 48% , and PBCA-bFGF-NP with 120.5nm of diameter, 89.35%of entrapment efficiency. And the experiment design method we used in these three prescription selecting we compared, the last composed design seemed more alluring than others.
Drug release test in vitro indicated that the drug release law of PBCA-bFGF-NP accorded with double-phase kinetical equation and Polynomial equation, and had obvious sustained-release specific property compared with the original drug. The observation to the reserving sample maintained frozen for three months showed that the stability of PBCA-bFGF-NP was bad, but the nanoparticle physicochemical properties had no evident difference.
To detect the biological activity of PBCA-bFGF-NP in vitro, chorioallantoic membrane (CAM) test were employed. The nanoparticles of the high and middle-dose groups had the same biological activity as bFGF did. At the meanwhile, there is obvious discrepancy between high-dose group and low-dose group.
According to all the above studies, Application new type high polymer material, and the preparation techniques of nanoparticle, we obtain PBCA-bFGF-NP that have sustained-release effect. bFGF nanoparticle study indicated that using nanoparticle as vector in injection drug delivery system is feasible. It has fairly high academic significance and special prospect in the administration peptide and protein.
冠心病是目前人类死亡的主要疾病之一,在我国其发病率呈逐年增高趋势,越来越多医学工作者在不断寻求新的冠心病治疗方法,而治疗性血管生成(therapeutic angiogenesis)或称“生物搭桥”(biologic bypass)的方法是目前冠心病治疗研究的热点。近年来,随着分子生物学的发展,人们发现血管新生过程中,有许多细胞因子的参与,而碱性成纤维生长因子(basic fibroblast growth factors, bFGF)就是其中之一。bFGF作为成纤维细胞生长因子(fibroblast growth factors, FGF)中的一种类型,发挥着它在各方面的功效。外源性bFGF能够促进细胞分裂、趋化,诱导胚胎正常发育、创面愈合、神经再生及功能恢复和促进血管生长。但是bFGF在体内的半衰期为3～10分钟,全身应用将被快速灭活而难以发挥其有效作用;在溶液中的稳定性较差,局部穿刺分次给药也难以产生有效作用,并且不适合长期给药。因此研究bFGF的纳米粒对长期维持局部药物的有效浓度,促进新生血管生长有着重要意义。
本研究以碱性成纤维生长因子(bFGF)为模型药物,系统地研究了bFGF纳米粒的制备处方和工艺,考察了制剂的粒径及分布、包封率及载药量和体外释药的特点,并对药物的体外活性进行了初步的研究。
利用酶联免疫吸附法(Enzyme Linked Immunosorbent Assay,ELISA)对碱性成纤维生长因子的含量进行测定,研究了碱性成纤维生长因子在制备条件下的稳定性。结果表明:药物在冷冻条件下保存最稳定,至少可保存一个月不降解;冷藏条件下保存,7天后药物浓度有所下降,药物活性部分丧失,故该药制成品宜冷冻保存。在4～15℃范围内、pH值在2.0～7.0之间以及搅拌速度为880r?min-1搅拌2h的条件下,制备成的纳米粒不会影响碱性成纤维生长因子的生物活性。
以粒径及其分布和包封率为指标,比较了三种载体材料制备工艺上的差异以及在质量上的区别,由实验结果可得出,以α-氰基丙烯酸正丁酯为载体的纳米粒包封率最高、制备方法简单、耗时最短,从而筛选出本研究所采用的载体材料α-氰基丙烯酸正丁酯。同时还得到得到粒径为89.9nm,包封率为28.13%的PLA-bFGF-NP和粒径为453nm,包封率为48%的CS-bFGF-NP。单因素实验考察了表面活性剂的种类和用量、载体材料的用量、药物与材料比例、pH值、搅拌速度等对碱性成纤维生长因子的影响,在单因素实验考察基础上,采用正交设计优化处方和制备工艺,得到粒径为120.5nm,包封率为89.35%的PBCA-bFGF-NP。
α-氰基丙烯酸正丁酯纳米粒的体外释放实验中,2h内释放小于30%,没有突释效应,而释放70%需要3天左右,具有明显的缓释效果,胶体溶液的释放可用双指数双相动力学方程或多项式方程描述,相关系数为分别为0.9905和0.9947。
在体外活性实验中,以鸡胚尿囊膜( Chorioallantoic Membrane , CAM )为动物模型,以无菌生理盐水作为阴性对照组,bFGF原料药为阳性对照组,分别比较高、中、低剂量PBCA-bFGF-NP对CAM血管增生的影响。实验结果显示:PBCA-bFGF-NP能够促进血管增生;统计结果表明:高、中剂量组与阴性对照组、低剂量组有明显的差异,而高、中剂量组和阳性对照组没有明显的差异。
本研究通过应用新型高分子材料和纳米粒制备技术,得到PBCA-bFGF-NP,其缓释效果达到项目设计要求。研究工作丰富了蛋白多肽类药物的缓释给药系统研究内容,具有较大的理论意义和广阔前景,具有较好的应用开发价值。
With the booming development and widely application of polypeptide and protein as an important method using in clinic, more and more urgent request are put through for a new drug delivery system of this kind of medicine instead of traditional pharmaceutical dosage form. To overcome the disadvantage of traditional form of medication, such as the pain has to suffer and the expensive fees in the curing process, a new drug new drug delivery system has to be found. It is necessary and full of esperance for both the medicine society and the pharmaceutical manufactures.
Nanoparticle has been regarded as a useful vector for targeted drug delivering system recent years. Many people pay attention to on the nanoparticles formed of biodegraded materials, such as polylactide (PLA), chitosan and butyl-2-cyanoacrylate (BCA), et al. We use them to protect drug form being degraded and delivery the drug into blood circle.
The basic fibroblast growth factors (bFGF) is a type of the fibroblast growth factors (FGF), it brought effect in many fieds. The ectogenesis basic fibroblast growth factors can promote the cell division and chemotaxis, and induce the normal growth of embryo, and promote the growth of blood vessel, create to regeneration of nerve and healing of wound. But the half-life of bFGF is about 3-5 minutes in bodys. If it is applied to whole body ,it is quickly caused to inactivation and hard to develope its valid function. The bFGF is unstability in solutions. It is very difficult to effect that it is applied many times though local puncture, and bFGF is not suitable for over a long period of time to the medicine. So it is very important significance that to study on sustained release bFGF nanoparticle of maintenancing the local effect of drug and promoting the the growth of blood vessel.
By using enzyme linked immunosorbent assay (ELISA) method , we studied the effect of different temperature、pH value and the rotation speed upon bFGF under the preparation condition. The optimum maintaining condition is to be preserved by freezing. No matter bathed in 37℃water for 24 hours or kept in tri-hydroxyl aminomethane buffer solution (Tris) (pH2.0) under 15℃for 1 hours, bFGF can not degraded. And the rotation speed of 880 revolutions per minute for 2 hours is safe for the preparation. The experiment indicates that bFGF is stable enough to survive through the whole preparation process.
The effects of different concentration of surfactant and polymer, the drug: polymer ratio and the pH value in the nanoparticle forming process were investigated. All these factors influence the size and entrapment efficiency obviously. Respectively, we use three kinds of method to optimize the formulation of the prescription and obtained PLA-bFGF-NP, which diameter is 89.9 nm and EE is28.13% , CS-bFGF-NP that diameter is 453nm and EE is 48% , and PBCA-bFGF-NP with 120.5nm of diameter, 89.35%of entrapment efficiency. And the experiment design method we used in these three prescription selecting we compared, the last composed design seemed more alluring than others.
Drug release test in vitro indicated that the drug release law of PBCA-bFGF-NP accorded with double-phase kinetical equation and Polynomial equation, and had obvious sustained-release specific property compared with the original drug. The observation to the reserving sample maintained frozen for three months showed that the stability of PBCA-bFGF-NP was bad, but the nanoparticle physicochemical properties had no evident difference.
To detect the biological activity of PBCA-bFGF-NP in vitro, chorioallantoic membrane (CAM) test were employed. The nanoparticles of the high and middle-dose groups had the same biological activity as bFGF did. At the meanwhile, there is obvious discrepancy between high-dose group and low-dose group.
According to all the above studies, Application new type high polymer material, and the preparation techniques of nanoparticle, we obtain PBCA-bFGF-NP that have sustained-release effect. bFGF nanoparticle study indicated that using nanoparticle as vector in injection drug delivery system is feasible. It has fairly high academic significance and special prospect in the administration peptide and protein.
引文
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[1] 李劲松,赵涵芳,陈诗书.bFGF 及其受体的研究进展[J].上海免疫学杂志,2002,22(5):357-360.
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[13] HASEGAWA T,KIMURA A, MIYATAKA M, et al. Basic fibroblast growth factor increases regional myocardial blood flow and salvages myocardium in the infarct border zone in a rabbit model of acute myocardial infarction .J. Angiology, 1999, 50 (6): 487-492.
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[15] Iwatate M, Miura T, Ikeda Y, et al. Effects of in vivo gene transfer of fibroblast growth factor-2 on cardiac function and collateral vessel formation in the microembolized rabbit heart. J. Jpn Circ 2001,65: 226-231.
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[17] 李易,马雁冰,孙林,等.rhbFGF 对缩小兔急性心肌梗死面积作用的观察[J].中国循环杂志,2002,17(3):232.
[18] 孙林,光雪峰,李易,等.碱性成纤维细胞生长因子基因对猪缺血心肌侧支血管生成的作用[J].中国介入心脏病学杂志,2006,14(3):188-190.
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[22] Ruel M, Laham RJ, Parker JA, et al. Long-term effects of surgical angiogenic therapy with fibroblast factor 2 protein. J.Thorac Cardiovasc Surg, 2002, 124: 28-34.
[23] Chen C, Li J , Mattar SG , et al. Boundary layer infusion of basic fibroblast growth factor accelerates intimal hyperplasia in endarterectomized canine artery. J. Surg Res, 1997, 69: 300-306.
[24] 乔延国,刘辉,王发强,等.碱性成纤维细胞生长因子预防球囊成形血管再狭窄[J].临床心血管病杂志,2000,16 (4):180-182.