胸腺五肽脂质体的研究
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摘要
脂质体是一种新型的药物载体,因其具有高度的生物靶向性、长效缓释性和低毒性引起了人们的广泛关注,在药物研究开发的许多领域中得到应用,并取得了很大进展。
本研究以胸腺五肽为模型药物,系统地研究了胸腺五肽脂质体的制备处方。本文在预实验的基础上,选取逆向蒸发法制备脂质体,初步研究了空白脂质体的制备工艺并进行了工艺优化和稳定性考察,结合粒径和包封率等因素优选出最优处方。在此基础上,制备了胸腺五肽脂质体,发现添加维生素E可以提高胸腺五肽的稳定性:最优处方制备的胸腺五肽脂质体的平均粒径为1.1012μm,包封率为64.8%。为了延长贮存期,对脂质体进行冷冻干燥处理,增加其体外稳定性。复溶后,显微观察到完整脂质体结构。
用紫外分光光度计法测定不同温度和超声条件对胸腺五肽造成的影响,结果表明:溶液冷藏4℃保存4天后开始降解,在37℃、0.1M pH7.0 PBS缓冲液中24小时保持稳定,超声功率在200~500W范围内,超声5分钟TP5活性还在。所以在制备胸腺五肽脂质体时,水浴25℃,pH7.0左右,300W超声5分钟,在1小时内完成制备不会破坏胸腺五肽。
用体内药效学研究来评价脂质体时,发现灌胃胸腺五肽脂质体对免疫缺损小鼠的白细胞数目、CD3+和CD4+数目百分比疗效与静脉注射胸腺五肽的药效无显著性差异。提示口服所制备的胸腺五肽脂质体能替代胸腺五肽注射剂在体内产生提升免疫力的生物效应。
本研究结果表明,虽然脂质体的稳定性有待提高,但以脂质体为载体包裹肽类药物通过口服途径给药,仍有较好的应用开发价值。
Liposome has been accepted into the family of drug delivery system recently with many advantages such as targeting specificity, sustained-release and low level of toxicity in biological systems. It has aroused a widespreaded interest and has many applications in the field of drug research.
After a systematic evaluation, a reverse evaporation method was selected to prepare liposome vesicles, which was then used as blank and used in the studies of optimization and stability. The particle size and encapsulation efficacy were the factors considered for optimal prescription of Thymopentin (TP5) liposome. The final thymopentin liposome was found to have partical size of 1.1μm, and the encapsulation rate of 64.8%. Adding vitamin E and freeze-drying were found to improve the stability of thymopentin liposome in vitro. Microscopic observation on the dissoluted freeze-dried TP5 liposome showed a structural integrity.
The results of UV evaluation on the concentration changes of TP5 during the prepartion process found no change in TP5 concentration under the conditions of pH 7.0 for 24 hours, or under the ultrasonic power of 200~500W within five minutes. TP5 liposome was found to be stable at 4℃for four days or 37℃for 24 hours in 0.1M PBS pH 7.0 buffer solution. Therefore, the preparation process under the condition of 25°C water bath, pH 7.0, ultrasonic power 300W for five minutes, and to complete the entire process within one hour will not disrupt thymopentin within liposome.
In vivo studies indicated that the therapeutic efficacy in terms of leukocyte and percentage of CD3+ and CD4+ in immune defected mice was no significant difference among the group receiving thymopentin liposomes given by gavage ,the group receiving thymopentin given by peritoneal injections , or the group receiving thymopentin given by intra venous injections. This result suggested that the TP5 liposomes prepared was a bioactive formulation.
Although the stability of the liposomes still needs to be improved further, our study implicated a favorable prospect in the application of liposomes as oral peptide delivery system.
本研究以胸腺五肽为模型药物,系统地研究了胸腺五肽脂质体的制备处方。本文在预实验的基础上,选取逆向蒸发法制备脂质体,初步研究了空白脂质体的制备工艺并进行了工艺优化和稳定性考察,结合粒径和包封率等因素优选出最优处方。在此基础上,制备了胸腺五肽脂质体,发现添加维生素E可以提高胸腺五肽的稳定性:最优处方制备的胸腺五肽脂质体的平均粒径为1.1012μm,包封率为64.8%。为了延长贮存期,对脂质体进行冷冻干燥处理,增加其体外稳定性。复溶后,显微观察到完整脂质体结构。
用紫外分光光度计法测定不同温度和超声条件对胸腺五肽造成的影响,结果表明:溶液冷藏4℃保存4天后开始降解,在37℃、0.1M pH7.0 PBS缓冲液中24小时保持稳定,超声功率在200~500W范围内,超声5分钟TP5活性还在。所以在制备胸腺五肽脂质体时,水浴25℃,pH7.0左右,300W超声5分钟,在1小时内完成制备不会破坏胸腺五肽。
用体内药效学研究来评价脂质体时,发现灌胃胸腺五肽脂质体对免疫缺损小鼠的白细胞数目、CD3+和CD4+数目百分比疗效与静脉注射胸腺五肽的药效无显著性差异。提示口服所制备的胸腺五肽脂质体能替代胸腺五肽注射剂在体内产生提升免疫力的生物效应。
本研究结果表明,虽然脂质体的稳定性有待提高,但以脂质体为载体包裹肽类药物通过口服途径给药,仍有较好的应用开发价值。
Liposome has been accepted into the family of drug delivery system recently with many advantages such as targeting specificity, sustained-release and low level of toxicity in biological systems. It has aroused a widespreaded interest and has many applications in the field of drug research.
After a systematic evaluation, a reverse evaporation method was selected to prepare liposome vesicles, which was then used as blank and used in the studies of optimization and stability. The particle size and encapsulation efficacy were the factors considered for optimal prescription of Thymopentin (TP5) liposome. The final thymopentin liposome was found to have partical size of 1.1μm, and the encapsulation rate of 64.8%. Adding vitamin E and freeze-drying were found to improve the stability of thymopentin liposome in vitro. Microscopic observation on the dissoluted freeze-dried TP5 liposome showed a structural integrity.
The results of UV evaluation on the concentration changes of TP5 during the prepartion process found no change in TP5 concentration under the conditions of pH 7.0 for 24 hours, or under the ultrasonic power of 200~500W within five minutes. TP5 liposome was found to be stable at 4℃for four days or 37℃for 24 hours in 0.1M PBS pH 7.0 buffer solution. Therefore, the preparation process under the condition of 25°C water bath, pH 7.0, ultrasonic power 300W for five minutes, and to complete the entire process within one hour will not disrupt thymopentin within liposome.
In vivo studies indicated that the therapeutic efficacy in terms of leukocyte and percentage of CD3+ and CD4+ in immune defected mice was no significant difference among the group receiving thymopentin liposomes given by gavage ,the group receiving thymopentin given by peritoneal injections , or the group receiving thymopentin given by intra venous injections. This result suggested that the TP5 liposomes prepared was a bioactive formulation.
Although the stability of the liposomes still needs to be improved further, our study implicated a favorable prospect in the application of liposomes as oral peptide delivery system.
引文
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