壳低聚糖及氨基葡萄糖与角蛋白相互作用的体外研究
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摘要
透皮给药系统具有明显优于其他传统给药方式的特点,市场应用前景广阔。然而皮肤角质层的屏障作用限制了大多数药物在透皮给药中的应用。壳低聚糖类化合物具有高效无毒的生理活性,已有相关研究表明能够有效促进药物的透皮吸收。本论文以人发提取角蛋白和脱脂处理的鼠角质层作为模拟皮肤的屏障模型,分别研究了壳低聚糖、氨基葡萄糖及氨基葡萄糖烷基酰胺类化合物对皮肤角质层的作用规律,为进一步研究糖类透皮促进剂的作用机制奠定基础。
本文首先采用以β-巯基乙醇为还原剂,尿素为蛋白质变性剂,十二烷基硫酸钠为蛋白质稳定剂的混合体系,自人发中提取角蛋白。实验发现反应前溶胀人发2h,选定尿素浓度为7mol/L,β-巯基乙醇和SDS的加入量分别在3.5%和2%,在45℃下反应12h,人发溶解率已经达到较高程度,制得的角蛋白溶液比较稳定。角蛋白膜不溶于THF、DMF、DMSO等常规有机溶剂,但在80℃热水中有溶胀现象,并可溶于低浓度的β-巯基乙醇水溶液(≥0.2wt %)。人发提取角蛋白膜与Sigma标准角蛋白膜对照,均属于致密膜,表面分布比较均匀。13C NMR谱图结果显示,人发提取角蛋白与鼠角蛋白和胶原结构基本相似,说明这种角蛋白可用来模拟动物皮肤角质层主要成分角蛋白,以此作为考察壳聚糖类促进剂相互作用的体外透皮模型。
同时,本论文还通过SEM,FTIR,DSC及电化学交流阻抗等手段探讨了促进剂对两种体外透皮模型的作用机理。浓度为1%的壳低聚糖和氨基葡萄糖都明显改变了角蛋白表面致密均匀的结构,并使角蛋白二级结构中α-螺旋显著地向β-折叠结构和无规则卷曲结构转变。角蛋白膜的α-helix结晶熔融峰为201℃左右,5%的壳低聚糖和氨基葡萄糖则分别使其结晶熔点下降至189℃和165℃,影响了角蛋白的结晶结构。5%的壳低聚糖使鼠脱脂角质层交流阻抗值由3.79E6?·cm2下降至8.379E5?·cm2,而氨基葡萄糖烷基酰胺DGP和DGH的这一数值分别降至1.001E5?·cm2和1.772E5?·cm2。角质层经皮阻抗值降低较为明显,表明两种糖类化合物有效降低了鼠角质层结构的紧密程度,为药物的经皮吸收与传送提供了通道。
Transdermal drug delivery system (TDDS) has much more advantages than other traditional modes, the application of which is an area of growing potential and development in pharmaceutical industry. However, the skin barrier limits the development of many drugs in TDDS. Chitosan oligomers(CS) and its relative derivatives, for its safe and effective properties, have been shown having the ability to aid the drugs delivery through skin. In this thesis, keratin from human hair(KH) and mouse stratum corneum(SC) were chose as in vitro models to research the effects and mechanism of CS, D-glucosamine (DG), DGP and DGH in the TDDS.
The project of dissolving KH with the mixed solvent system of reducer, denature reagent and surfactants was firstly confirmed, which containedβ-mercaptoethanol, Urea and SDS, respectively. The results showed that when the hair was reduced with the mixture respectively being 3.5%, 7mol/L and 2% at 45°C for 12h, a steady keratin solution was gained while of a higher hair reducing ratio. Keratin membrane was obtained insoluble in normal organic solvent, such as THF, DMF, DMSO, could slightly swell in hot water(80°C), but easily dissolved inβ-mercaptoethanol solution (≥0.2%). Both of the KH membrane and keratin from Sigma were dense and homogeneous. 13C NMR researched on KH showed that which had resemble structure of mouse SC and collagen. So KH can be used as a model in vitro for researching on the effects of CS and its relative derivatives in TDDS.
Meanwhile, SEM, FTIR, DSC and EIS were used to study the mechanism of transdermal enhancers in the vitro models. Both CS and DG with the concentration of 1% changed the dense and homogeneous on keratin surface, while the secondary structure ofα-helix clearly shifted toβ-sheet in relation to the untreated keratin sample. The enhancers with the concentration of 5% decreased the melting of a-form crystallites in KH while the endothermal peak temperature reduced from 201°C to 189°C and 165°C, respectively. The EIS values of Mouse SC reduced from 3.79E6?·cm2 to 8.379E5?·cm2, 1.001E5 ?·cm2 and 1.772E5 ?·cm2 after treatment with CS, DGP and DGH, respectively. The results showed that the application of CS and its relative derivatives to confluent keratin monolayers can result in reduction of trans-epithelial electrical resistance(TEER) indicating an effect on tight junctions, thus the enhancers aid the drug permeation into the SC.
本文首先采用以β-巯基乙醇为还原剂,尿素为蛋白质变性剂,十二烷基硫酸钠为蛋白质稳定剂的混合体系,自人发中提取角蛋白。实验发现反应前溶胀人发2h,选定尿素浓度为7mol/L,β-巯基乙醇和SDS的加入量分别在3.5%和2%,在45℃下反应12h,人发溶解率已经达到较高程度,制得的角蛋白溶液比较稳定。角蛋白膜不溶于THF、DMF、DMSO等常规有机溶剂,但在80℃热水中有溶胀现象,并可溶于低浓度的β-巯基乙醇水溶液(≥0.2wt %)。人发提取角蛋白膜与Sigma标准角蛋白膜对照,均属于致密膜,表面分布比较均匀。13C NMR谱图结果显示,人发提取角蛋白与鼠角蛋白和胶原结构基本相似,说明这种角蛋白可用来模拟动物皮肤角质层主要成分角蛋白,以此作为考察壳聚糖类促进剂相互作用的体外透皮模型。
同时,本论文还通过SEM,FTIR,DSC及电化学交流阻抗等手段探讨了促进剂对两种体外透皮模型的作用机理。浓度为1%的壳低聚糖和氨基葡萄糖都明显改变了角蛋白表面致密均匀的结构,并使角蛋白二级结构中α-螺旋显著地向β-折叠结构和无规则卷曲结构转变。角蛋白膜的α-helix结晶熔融峰为201℃左右,5%的壳低聚糖和氨基葡萄糖则分别使其结晶熔点下降至189℃和165℃,影响了角蛋白的结晶结构。5%的壳低聚糖使鼠脱脂角质层交流阻抗值由3.79E6?·cm2下降至8.379E5?·cm2,而氨基葡萄糖烷基酰胺DGP和DGH的这一数值分别降至1.001E5?·cm2和1.772E5?·cm2。角质层经皮阻抗值降低较为明显,表明两种糖类化合物有效降低了鼠角质层结构的紧密程度,为药物的经皮吸收与传送提供了通道。
Transdermal drug delivery system (TDDS) has much more advantages than other traditional modes, the application of which is an area of growing potential and development in pharmaceutical industry. However, the skin barrier limits the development of many drugs in TDDS. Chitosan oligomers(CS) and its relative derivatives, for its safe and effective properties, have been shown having the ability to aid the drugs delivery through skin. In this thesis, keratin from human hair(KH) and mouse stratum corneum(SC) were chose as in vitro models to research the effects and mechanism of CS, D-glucosamine (DG), DGP and DGH in the TDDS.
The project of dissolving KH with the mixed solvent system of reducer, denature reagent and surfactants was firstly confirmed, which containedβ-mercaptoethanol, Urea and SDS, respectively. The results showed that when the hair was reduced with the mixture respectively being 3.5%, 7mol/L and 2% at 45°C for 12h, a steady keratin solution was gained while of a higher hair reducing ratio. Keratin membrane was obtained insoluble in normal organic solvent, such as THF, DMF, DMSO, could slightly swell in hot water(80°C), but easily dissolved inβ-mercaptoethanol solution (≥0.2%). Both of the KH membrane and keratin from Sigma were dense and homogeneous. 13C NMR researched on KH showed that which had resemble structure of mouse SC and collagen. So KH can be used as a model in vitro for researching on the effects of CS and its relative derivatives in TDDS.
Meanwhile, SEM, FTIR, DSC and EIS were used to study the mechanism of transdermal enhancers in the vitro models. Both CS and DG with the concentration of 1% changed the dense and homogeneous on keratin surface, while the secondary structure ofα-helix clearly shifted toβ-sheet in relation to the untreated keratin sample. The enhancers with the concentration of 5% decreased the melting of a-form crystallites in KH while the endothermal peak temperature reduced from 201°C to 189°C and 165°C, respectively. The EIS values of Mouse SC reduced from 3.79E6?·cm2 to 8.379E5?·cm2, 1.001E5 ?·cm2 and 1.772E5 ?·cm2 after treatment with CS, DGP and DGH, respectively. The results showed that the application of CS and its relative derivatives to confluent keratin monolayers can result in reduction of trans-epithelial electrical resistance(TEER) indicating an effect on tight junctions, thus the enhancers aid the drug permeation into the SC.
引文
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[4]孙耀华,药剂学,北京:人民卫生出版社, 2003, 263～264
[5]梁秉文,经皮给药制剂,北京:中国医药科技出版社, 1992
[6]郑俊民,经皮给药新剂型,北京:人民卫生出版社, 1997, 36~56
[7] Purdon C H, Azzi C G, Zhang J, et al. Penetration enhancement of transdermal delivery-current permutations and limitations, Critical Reviews in Therapeutic Drug Carrier Systems, 2004, 21(2): 97~132
[8] Bouwstra J, Transdermal drug administration, http://srdweb2.dl.ac.uk/srs/, 2002
[9]韩国柱,中草药药代动力学,北京:中国医药科技出版社, 1998, 112~115
[10] Marjukka S T, Joke A, Bouwstra, et al. Chemical enhancement of percutaneous absorption in relation to stratum corneum structural alterations, Journal of Controlled Release, 1999, 59:149~161
[11] Flynn G L , Topical drug absorption and topical pharmaceutical systems, in: G.S. Banker, C.T. Rhodes (Eds.), Modem Pharmaceutics, Marcel Dekker, New York, 1990, pp.263~325
[12] Hueber F, Schaefer H, Wepierre J, Role of transepidermal and transfollicular routes in percutaneous absorption of steroids: in vitro studies on human skin, Skin Pharmacol, 1994,7:237~244
[13] Hadgraft J, Transdermal drug delivery: development issue and research initiatives, New York: Marcel Dekker, Inc, 1988, 1～16
[14] Asbill, Charles S, Michniak, et al. Percutaneous penetration enhancers: local versus transdermal activity, Pharma. Sci. Technol. Today, 2000, 3(1): 36～41
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