酸性成纤维细胞生长因子阳离子脂质体的研制及其在大鼠体内药动学研究
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摘要
aFGF具有多种生物学活性,但由于稳定性差,对温度、pH以及金属离子敏感,极易失活等众多缺点,极大限制了其临床应用的范围。
目的：以阳离子脂质体作为药物aFGF的载体,以实现制剂对aFGF的有效保护作用和制剂缓释作用为目标,研究制备工艺,建立其质量评价体系,并进行体外释药动力学及大鼠体内药动学研究。
方法：①pH梯度法制备aFGF阳离子脂质体,以包封率为考察指标单因素筛选aFGF阳离子脂质体最佳制备工艺,并考察该工艺的重现性。②采用低温电镜和激光散射法测定aFGF阳离子脂质体的理化性质,并进行了稳定性考察。③采用透析法研究aFGF阳离子脂质体体外释药动力学,考察其与aFGF溶液组的差别及释放介质对其释药的影响。④从静脉与腹腔注射两种给药方式,研究aFGF阳离子脂质体体内药动学,并观察aFGF溶液与aFGF脂质体药动学的差别。
结果：①筛选最佳制备工艺为：十八胺用量为0.02g,柠檬酸缓冲液浓度为0.03mol／L,aFGF投药量为0.04 mg/mL,内外水相ΔpH为3.5,孵育温度为35℃,孵育时间为15 min,制备的aFGF阳离子脂质体包封率为(78.82±2.56)%。②aFGF阳离子脂质体平均粒径为124.03 nm,ζ电位为9.68 mV,在温度25℃以上不稳定,宜低温4℃保存。③体外释药动力学研究结果表明,与aFGF原液相比,aFGF阳离子脂质体,在不同释放介质中均具有一定的缓释作用。④无论腹腔还是静脉注射,药动学结果表明,aFGF阳离子脂质体明显延长了体内半衰期,且与体外释药存在良好相关性。
结论：制备出一种aFGF阳离子脂质体,具有包封率高,理化性质稳定,及显著的体内缓释作用。
As we know aFGF (acid fibroblast growth factor) plays an important role in organism for its numerous bioactivity.However, aFGF is unstable and sensitive to temperature, pH and metal ion, it still has a bottle neck on clinical application.
Objective: In this study, we made cationic liposome as a drug carrier for aFGF in order to effectively protect aFGF from unstabilizing factors, established the quality assessment system of aFGF cationic liposome,and researched on its release kinetics in vitro and pharmacokinetic in vivo.
Methods:AFGF cationic liposomes were prepared by pH-gradient, the formulation and preparation were optimized by single-factor test with encapsulation efficiency of liposomes as the parameter, and the reproducibility of the best preparation was studied.The physical and chemistry properties of aFGF cationic liposome was identified by Cryo transmission electron microscopy and laser light scattering, and its stability was also researched. The vitro release kinetics of aFGF cationic liposome was investigated by Dialysis method. Observed the influence of drug release between different release medium, and also researched the difference with aFGF solution. Studied pharmacokinetic of aFGF cationic liposomes after intravenous and intraperitoneal administration in rats, and discussed the difference with aFGF solution.
Results:Optimization experiments to determine the optimal formulation and process conditions, stearylamine (0.02 g), citric acid buffer (0.03 mol/L), aFGF (0.04 mg/mL), ApH (3.5), incubation temperature (35℃), incubation time (15 min), the preparation of aFGF cationic liposome encapsulation efficiency was (78.82±2.56)%.The mean diameter of aFGF cationic liposomes was 124.03nm, Zeta potential was 9.68mV. Stability studies indicated that aFGF liposome should be stored at low temperature 4℃, it was unstable above 25℃.In vitro release kinetics study showed that, aFGF cationic liposome had some sustained release compared to aFGF solution, similar behavior in various release medium.Pharmacokinetic results had showned that aFGF cationic liposomes obvious prolonged the half-life in vivo, compared with aFGF solution. And there is a good correlation between the release characters in vivtro with the release in vitro.
Conclusion:Research indiated, aFGF cationic liposomes had high encapsulating efficiency, physical chemical stability, and sustained release in vivo.
目的：以阳离子脂质体作为药物aFGF的载体,以实现制剂对aFGF的有效保护作用和制剂缓释作用为目标,研究制备工艺,建立其质量评价体系,并进行体外释药动力学及大鼠体内药动学研究。
方法：①pH梯度法制备aFGF阳离子脂质体,以包封率为考察指标单因素筛选aFGF阳离子脂质体最佳制备工艺,并考察该工艺的重现性。②采用低温电镜和激光散射法测定aFGF阳离子脂质体的理化性质,并进行了稳定性考察。③采用透析法研究aFGF阳离子脂质体体外释药动力学,考察其与aFGF溶液组的差别及释放介质对其释药的影响。④从静脉与腹腔注射两种给药方式,研究aFGF阳离子脂质体体内药动学,并观察aFGF溶液与aFGF脂质体药动学的差别。
结果：①筛选最佳制备工艺为：十八胺用量为0.02g,柠檬酸缓冲液浓度为0.03mol／L,aFGF投药量为0.04 mg/mL,内外水相ΔpH为3.5,孵育温度为35℃,孵育时间为15 min,制备的aFGF阳离子脂质体包封率为(78.82±2.56)%。②aFGF阳离子脂质体平均粒径为124.03 nm,ζ电位为9.68 mV,在温度25℃以上不稳定,宜低温4℃保存。③体外释药动力学研究结果表明,与aFGF原液相比,aFGF阳离子脂质体,在不同释放介质中均具有一定的缓释作用。④无论腹腔还是静脉注射,药动学结果表明,aFGF阳离子脂质体明显延长了体内半衰期,且与体外释药存在良好相关性。
结论：制备出一种aFGF阳离子脂质体,具有包封率高,理化性质稳定,及显著的体内缓释作用。
As we know aFGF (acid fibroblast growth factor) plays an important role in organism for its numerous bioactivity.However, aFGF is unstable and sensitive to temperature, pH and metal ion, it still has a bottle neck on clinical application.
Objective: In this study, we made cationic liposome as a drug carrier for aFGF in order to effectively protect aFGF from unstabilizing factors, established the quality assessment system of aFGF cationic liposome,and researched on its release kinetics in vitro and pharmacokinetic in vivo.
Methods:AFGF cationic liposomes were prepared by pH-gradient, the formulation and preparation were optimized by single-factor test with encapsulation efficiency of liposomes as the parameter, and the reproducibility of the best preparation was studied.The physical and chemistry properties of aFGF cationic liposome was identified by Cryo transmission electron microscopy and laser light scattering, and its stability was also researched. The vitro release kinetics of aFGF cationic liposome was investigated by Dialysis method. Observed the influence of drug release between different release medium, and also researched the difference with aFGF solution. Studied pharmacokinetic of aFGF cationic liposomes after intravenous and intraperitoneal administration in rats, and discussed the difference with aFGF solution.
Results:Optimization experiments to determine the optimal formulation and process conditions, stearylamine (0.02 g), citric acid buffer (0.03 mol/L), aFGF (0.04 mg/mL), ApH (3.5), incubation temperature (35℃), incubation time (15 min), the preparation of aFGF cationic liposome encapsulation efficiency was (78.82±2.56)%.The mean diameter of aFGF cationic liposomes was 124.03nm, Zeta potential was 9.68mV. Stability studies indicated that aFGF liposome should be stored at low temperature 4℃, it was unstable above 25℃.In vitro release kinetics study showed that, aFGF cationic liposome had some sustained release compared to aFGF solution, similar behavior in various release medium.Pharmacokinetic results had showned that aFGF cationic liposomes obvious prolonged the half-life in vivo, compared with aFGF solution. And there is a good correlation between the release characters in vivtro with the release in vitro.
Conclusion:Research indiated, aFGF cationic liposomes had high encapsulating efficiency, physical chemical stability, and sustained release in vivo.
引文
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[2]Yamashita T, Yoshioka M, Itoh N. Identifaction of a novel fibroblast growth factor, FGF-23, perferrntially expressed in the ventrolateral thalamis nucleus of the brain [J]. Biochim Biophys Res Commun,2000,277(2):494-498
[3]Pan Y, Dai F, Chen Y. DNA contents and cycle analysis of porcine burn wounds treated with growth factors [J]. Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi,1996,12 (4):262-264
[4]屠重棋,袁淑兰,王艳萍等.重组人酸性成纤维细胞生长因子对人成骨细胞生长及增殖的影响[J]中华骨科杂志,2001,21(8)：489-492
[5]张建军,陈书艳,张爱兵等.酸性成纤维细胞生长因子对微血管生成的影响[J].中华心血管病杂志,1998,26(2)：97
[6]郭艳芹,安丽荣,关亚新.aFGF对Aβ25-35诱导的PC12细胞凋亡的保护作用[J].中国老年学杂志,2008,2(28)：244-246
[7]连芳,黄巨恩,胡世凤等.酸性成纤维细胞生长因子对大鼠急性心肌缺血损伤的保护作用[J].广西医科大学学报,2008,25(5)：672-673
[8]Hua Xu, Guang-fan Hai,Ji-zhou Xiang,et al. Protective effect of non-mitogenic human acidic fibroblast growth factor on hepatocyte injury[J].Hepatology Research,2007,37(10):836-844
[9]Xu H, Hai GF, Xu H, et al. Protective effect of non-mitogenic human acidic fibroblast growth factor from renal ischemia-reperfusion injury[J]. Chin J Pathophysiol,2008,24 (3):552-557.
[10]郑曙云,付小兵,徐建国等.酸性成纤维细胞生长因子对大鼠肠缺血再灌注损伤影响的研究[J].中国危重病急救医学.2004,16(8)：460-463
[11]陈伟,付小兵,葛世丽等.酸性成纤维细胞生长因子对缺血再灌注小肠绒毛细胞内bax和bcl-2表达的影响[J].世界华人消化杂志,2004,12(11)：2599-2604
[12]Zheng SY,Fu XB,Xu JG. Effects of acidic fibroblast growth factor on intestinal mucosal damage due to ischemia/reperfusion[J].Chin Crit CareMed,2004,16 (8):460-463
[13]Laird JMA, M ason GS, Thomas KA, et al. Acidic fibroblast growth factor stimulates motor and sensory axon regeneration after sciatic nervecrush in the rat [J].Neurosci,1995,65 (1):209
[14]Tada T, Ito J, Asai M, et al.Fibroblast growth factor is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain [J]. Neurochem Int,2004,45(1):23-30
[15]Thorns V,Licastro F,Masliah E. Locally reduced levels of acdic FGF lead to decreased expression of 28-kda calbindin and contribute to the selective vulnerability of the neurons in the entorhinal cortex in Alzheimer's disease[J].Neuropathology,2001,21(3):203-211
[16]Bangham AD, Home RW. Negative staining of phospholipidsand their structural modification by surface-active agents as observed in the electron microscope [J]. J Mol Biol,1964,8(12): 660-668
[17]Gregoriadis G, Leathwood PD, Ryman BE. Enzyme entrapment in liposomes [J]. FEBS Lett.1971,12(2):95-99
[18]Grit M, Crommelin DJA. The effect of surface charge on the hydrolysis kinetics of partially hydrogenated egg phosphatidylcholine and egg phosphatidylglycerol in aqueous liposome dispersions [J].Biochem Biophys Acta,1993,1167(1):49.
[19]Crommelin DJA. Influence of lipid composition and ionic strength on the physical stability of liposomes [J]. J Pharm Sci,1984,73(11):1559
[20]Hiromitsu Aoki, Tsuneaki Tottori, Fuminori Sakurai, et al. Effects of positive charge density on the liposomal surface on disposition kinetics of liposomes in rats [J]. International Journal of Pharmaceutics,1997,156(12):163-174
[21]郭秀侠,福泉,陈妍等.人用狂犬病毒脂质体疫苗的制备及性质[J].中国生物制品学杂志,2006,19(6)：589-591
[22]周臻,邓英杰.兰索拉唑脂质体的制备及性质考察[J].沈阳药科大学学报,2008,2(25)：81-84
[23]B ID Z1 Pharm aceutics(药剂学)[M].4 th Ed. Beijing: People's Health Publishing House, 2000:449-450
[24]Kellaway IW, Farr S J. Liposoms as drug delivery systems to the lung [J]. Adv Drug Dliv Rev, 1990,5 (1):149-161
[25]Caride VJ, Zaret BL.Liposomes accumulation in regions of experimental myocardial infarction [J].Science,1977,198(4318):735
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