基于超临界CO_2强化溶液分散法制备营养素脂质体前体的研究
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摘要
脂质体前体以固体形态存在,能有效地解决脂质体液态时易出现的聚集沉降、磷脂氧化水解以及包封药物泄漏等物化稳定性差的问题。它是一种带有有效成份的干燥的流动性好的颗粒,临用前分散于水中即可得到脂质体悬浮液。但是,目前常规的制备方法还存在稳定性差、制备耗时、溶剂残留等问题,无法满足工业生产需要。超临界CO_2强化溶液分散法不仅工艺简单,而且整个生产过程溶剂残留低,绿色环保无污染,在制备脂质体前体方面有巨大应用前景。目前,还没有超临界CO_2强化溶液分散法制备脂质体前体的系统研究,只有少量文献进行相关报道,对于超临界CO_2强化溶液分散法制备脂质体前体的影响因素的研究,制备过程中各因素的影响规律的研究仍很缺乏。
本课题利用超临界CO_2与有机溶剂互溶性强,制备的微粒粒度分布窄等特性,提出了超临界CO_2强化溶液分散法制备脂质体前体,然后水化脂质体前体制备脂质体的绿色工艺路线,构建了脂质体前体制备的绿色新工艺。建立了高压相平衡测定装置,为研究脂质体前体制备的影响因素与规律提供了理论依据。并对超临界抗溶剂法过程中涉及到的影响因素和规律进行了深入研究,为脂质体前体的工业化生产提供了理论依据和技术参数。对制备的脂质体前体进行了动物实验效果评价,为脂质体前体的临床应用提供了实验依据。主要结论如下:
1.通过测定CO_2+DCM+EtOH,CO_2+EtOH+C_6H_(14)以及HPC+EtOH+CO_2三种三元体系在不同温度和组成下的浊点和泡点压力,对超临界CO_2强化溶液分散法过程的相平衡进行研究。组成不变时,随着温度的升高,体系泡点压力增大;温度不变时,随着CO_2量的增大,体系泡点压力增大。极性较强的溶剂对体系相平衡的影响比极性较弱的溶剂要大。用PR-EOS方程成功对体系CO_2 +EtOH+ C_6H_(14)的相平衡数据进行了拟合。该研究为脂质体前体制备奠定了理论基础。
2.以辅酶Q10为辅酶类模型,粉末磷脂、胆固醇为壁材,考察了体系温度、压力、组成等对脂质体载药量的影响,用SEM, XRD, DLS和TEM对脂质体前体和脂质体进行了表征。确定了用超临界CO_2强化溶液分散法制备辅酶Q10脂质体前体的最佳条件:以体积比13:12的二氯甲烷和无水乙醇为混合溶剂,压力8.0MPa,温度35℃, CoQ10和PC质量比为1:10,胆固醇和PC的质量比为1:3。在此条件下,CoQ10载药量为8.92%。脂质体前体水化后可得到单分散粒径为50nm左右的脂质体悬浮液。说明通过超临界CO_2强化溶液分散法制备辅酶Q10脂质体前体,并通过脂质体前体水化得到脂质体的工艺路线可行。
3.以维生素D3为维生素模型药物,氢化磷脂为壁材,确定了最佳条件:压力8.0MPa,温度45℃, VD_3和HPC质量比为1.5:10, VD_3载药量为12.89%。在此条件下,VD_3载药量为12.89%,包埋率接近100%。通过与薄膜分散法制得的脂质体进行比较,发现脂质体前体水化后得到的脂质体粒径更小,粒度分布更均匀。说明该工艺路线相对常规方法薄膜分散法具有一定优势。通过SEM、TEM、XRD和DLS对脂质体前体和脂质体的表征,对脂质体前体水化得到脂质体的水化机理进行了研究,发现通过超临界CO_2强化溶液分散法得到的matrix结构对水化得到高包埋率的脂质体有促进作用。
4.以叶黄素为植物营养素模型药物,氢化磷脂为壁材,详细考察了实验条件对脂质体前体形貌、载药量的影响,确定了制备脂质体前体的最佳条件:压力8.0MPa,温度35℃,溶液流速1ml/min。在此条件下,叶黄素载药量为55mg/g,脂质体包埋率达90%以上,二氯甲烷溶剂残留量极低,为7.2ppb。通过动物实验发现,叶黄素脂质体的抗氧化活性较好,并且有明显的剂量依赖性。说明本文提出的将活性物制备成脂质体前体,临用前水化成脂质体的过程,对活性物的生物活性没有破坏,且溶剂残留量低,该工艺路线为脂质体前体工业化生产提供了新思路。
The concept of proliposomes is introduced to solve problems caused by the liquid form of liposomes, such as aggregation, fusion, hydrolysis of lipid, and so on. Proliposomes are defined as a kind of dry and free-flowing particles with loading ingredients. The liposome suspension can be easily obtained from proliposomes when they are dispersed in water. However, there are some shortcomings in the traditional methods: such as proper cryoprotectants needed, many steps involved, and high processing temperatures, which limit their wide application. To solve the problems above, solution enhanced dispersion by supercritical CO_2 widely used in food industry should be developed to prepare proliposomes, because of its lower residual solvents, simpler steps and mild operation temperatures. In the aspect of the preparation of proliposomes using supercritical CO_2 technique, limited papers are published. A few papers just involved in the preparation of phospholipid powders by supercriticalCO_2. Only one paper involved in the preparation of proliposomes containing miconazole by the aerosol solvent extraction system (ASES) process.
This research was to prepare proliposomes using solution enhanced dispersion by supercritical CO_2 (SEDS) and to make liposomes via the hydration of the proliposomes. Effects of the process parameters were investigated, and the animal experiments were introduced to determine the antioxidant activity of the liposomes. The purpose of our study was to provide the technical parameters and the theory basis for the industrialization of the liposomes. The conclusions were as follows:
1. Experimental data for the phase behavior of the four systems CO_2 + DCM + EtOH (ethanol), CO_2 + EtOH + C_6H_(14) (n-hexane) and HPC + EtOH + CO_2 (dichloromethane) with different compositions at temperatures from 308.5K to 328.5K were investigated. The bubble point pressure increases with increasing temperature at constant CO_2 mass fraction. In addition, the modeling results indicate that PR-EOS (Peng-Robinson equation of state) with one interaction parameter can correlate the experimental data for the bubble points of the system CO_2 + EtOH + C_6H_(14) (n-hexane).
2. The coenzyme Q10 was chosen as the model drug. The mixture of cholesterol and soybean phosphatidylcholine (PC) was chosen as wall materials. The effects of operation conditions (temperature, pressure and components) on the recovery of CoQ10 and the CoQ10 loading in CoQ10 proliposomes were studied. At the optimum conditions of pressure of 8.0MPa, temperature of 35℃, the weight ratio of 1/10 between CoQ10 and PC, and the weight ratio of 1/3 between cholesterol and PC, the CoQ10 loading reached 8.92%. CoQ10 liposomes were obtained by hydrating CoQ10 proliposomes and the entrapment efficiency of CoQ10 reached 82.28%. The morphology of CoQ10 proliposomes were characterized by SEM, and their solid state was characterized by XRD. The structure of CoQ10 liposomes were characterized by TEM. The particle size distribution of CoQ10 liposomes was determined by DLS. The results indicate that CoQ10 liposomes with particle sizes about 50nm can be easily got from hydrating CoQ10 proliposomes prepared by SEDS.
3. Vitamin D3 (VD_3) proliposomes, consisted of hydrogenated phosphatidycholine (HPC) and VD_3, were prepared using SEDS. The effects of operation conditions (temperature, pressure and components) on the VD_3 loading in VDP were studied. At the optimum conditions of pressure of 8.0MPa, temperature of 45℃, and the weight ratio of 15.0% between VD_3 and HPC, the VD_3 loading reached 12.89%. VD_3 liposomes were obtained by hydrating VD_3 proliposomes and the entrapment efficiency of VD_3 in VD_3 liposomes reached 98.5%. The morphology and structure of proliposomes and liposomes were characterized by SEM, TEM and XRD. The structure of VD_3 nanoparticles in HPC matrix was formed. The size of liposome was determined by Dynamic Light Scattering instrument (DLS). The average diameter of liposomes was about 1μm. The results indicate that VDP can be made by SEDS and liposomes with high entrapment efficiency can be formed easily via the hydration of proliposomes.
4. Proliposomes composed of lutein and hydrogenated phosphatidylcholine were prepared using SEDS. The effects of the process parameters on the lutein loading and the particle sizes of the proliposomes were investigated. HPLC was applied to determine the content of lutein in the samples. At the optimum conditions—temperature of 35℃, pressure of 8MPa and the solution flow rate of 1 ml/min—the lutein loading of the proliposomes reached 55mg/g. The images characterized by SEM were evaluated for the different proliposomes samples in order to study the influences of operational conditions on the particle sizes and morphology. When proliposome was hydrated, the lutein liposome suspensions were formed automatically. The crystallinity of proliposomes was analyzed using DSC to analyze the distribution of lutein in proliposomes. The structure of proliposomes and the lutein liposome was detected by TEM. The results indicate that proliposomes with the high lutein loading was made successfully and the lutein liposome was obtained with the encapsulation efficiency of more than 90% after hydrating proliposomes. The animal experiment results show that there is dose dependence on the lutein liposomes. These results demonstrate that SEDS technique is a simple and effective process for the preparation of proliposomes from which liposome can be easily formed.
本课题利用超临界CO_2与有机溶剂互溶性强,制备的微粒粒度分布窄等特性,提出了超临界CO_2强化溶液分散法制备脂质体前体,然后水化脂质体前体制备脂质体的绿色工艺路线,构建了脂质体前体制备的绿色新工艺。建立了高压相平衡测定装置,为研究脂质体前体制备的影响因素与规律提供了理论依据。并对超临界抗溶剂法过程中涉及到的影响因素和规律进行了深入研究,为脂质体前体的工业化生产提供了理论依据和技术参数。对制备的脂质体前体进行了动物实验效果评价,为脂质体前体的临床应用提供了实验依据。主要结论如下:
1.通过测定CO_2+DCM+EtOH,CO_2+EtOH+C_6H_(14)以及HPC+EtOH+CO_2三种三元体系在不同温度和组成下的浊点和泡点压力,对超临界CO_2强化溶液分散法过程的相平衡进行研究。组成不变时,随着温度的升高,体系泡点压力增大;温度不变时,随着CO_2量的增大,体系泡点压力增大。极性较强的溶剂对体系相平衡的影响比极性较弱的溶剂要大。用PR-EOS方程成功对体系CO_2 +EtOH+ C_6H_(14)的相平衡数据进行了拟合。该研究为脂质体前体制备奠定了理论基础。
2.以辅酶Q10为辅酶类模型,粉末磷脂、胆固醇为壁材,考察了体系温度、压力、组成等对脂质体载药量的影响,用SEM, XRD, DLS和TEM对脂质体前体和脂质体进行了表征。确定了用超临界CO_2强化溶液分散法制备辅酶Q10脂质体前体的最佳条件:以体积比13:12的二氯甲烷和无水乙醇为混合溶剂,压力8.0MPa,温度35℃, CoQ10和PC质量比为1:10,胆固醇和PC的质量比为1:3。在此条件下,CoQ10载药量为8.92%。脂质体前体水化后可得到单分散粒径为50nm左右的脂质体悬浮液。说明通过超临界CO_2强化溶液分散法制备辅酶Q10脂质体前体,并通过脂质体前体水化得到脂质体的工艺路线可行。
3.以维生素D3为维生素模型药物,氢化磷脂为壁材,确定了最佳条件:压力8.0MPa,温度45℃, VD_3和HPC质量比为1.5:10, VD_3载药量为12.89%。在此条件下,VD_3载药量为12.89%,包埋率接近100%。通过与薄膜分散法制得的脂质体进行比较,发现脂质体前体水化后得到的脂质体粒径更小,粒度分布更均匀。说明该工艺路线相对常规方法薄膜分散法具有一定优势。通过SEM、TEM、XRD和DLS对脂质体前体和脂质体的表征,对脂质体前体水化得到脂质体的水化机理进行了研究,发现通过超临界CO_2强化溶液分散法得到的matrix结构对水化得到高包埋率的脂质体有促进作用。
4.以叶黄素为植物营养素模型药物,氢化磷脂为壁材,详细考察了实验条件对脂质体前体形貌、载药量的影响,确定了制备脂质体前体的最佳条件:压力8.0MPa,温度35℃,溶液流速1ml/min。在此条件下,叶黄素载药量为55mg/g,脂质体包埋率达90%以上,二氯甲烷溶剂残留量极低,为7.2ppb。通过动物实验发现,叶黄素脂质体的抗氧化活性较好,并且有明显的剂量依赖性。说明本文提出的将活性物制备成脂质体前体,临用前水化成脂质体的过程,对活性物的生物活性没有破坏,且溶剂残留量低,该工艺路线为脂质体前体工业化生产提供了新思路。
The concept of proliposomes is introduced to solve problems caused by the liquid form of liposomes, such as aggregation, fusion, hydrolysis of lipid, and so on. Proliposomes are defined as a kind of dry and free-flowing particles with loading ingredients. The liposome suspension can be easily obtained from proliposomes when they are dispersed in water. However, there are some shortcomings in the traditional methods: such as proper cryoprotectants needed, many steps involved, and high processing temperatures, which limit their wide application. To solve the problems above, solution enhanced dispersion by supercritical CO_2 widely used in food industry should be developed to prepare proliposomes, because of its lower residual solvents, simpler steps and mild operation temperatures. In the aspect of the preparation of proliposomes using supercritical CO_2 technique, limited papers are published. A few papers just involved in the preparation of phospholipid powders by supercriticalCO_2. Only one paper involved in the preparation of proliposomes containing miconazole by the aerosol solvent extraction system (ASES) process.
This research was to prepare proliposomes using solution enhanced dispersion by supercritical CO_2 (SEDS) and to make liposomes via the hydration of the proliposomes. Effects of the process parameters were investigated, and the animal experiments were introduced to determine the antioxidant activity of the liposomes. The purpose of our study was to provide the technical parameters and the theory basis for the industrialization of the liposomes. The conclusions were as follows:
1. Experimental data for the phase behavior of the four systems CO_2 + DCM + EtOH (ethanol), CO_2 + EtOH + C_6H_(14) (n-hexane) and HPC + EtOH + CO_2 (dichloromethane) with different compositions at temperatures from 308.5K to 328.5K were investigated. The bubble point pressure increases with increasing temperature at constant CO_2 mass fraction. In addition, the modeling results indicate that PR-EOS (Peng-Robinson equation of state) with one interaction parameter can correlate the experimental data for the bubble points of the system CO_2 + EtOH + C_6H_(14) (n-hexane).
2. The coenzyme Q10 was chosen as the model drug. The mixture of cholesterol and soybean phosphatidylcholine (PC) was chosen as wall materials. The effects of operation conditions (temperature, pressure and components) on the recovery of CoQ10 and the CoQ10 loading in CoQ10 proliposomes were studied. At the optimum conditions of pressure of 8.0MPa, temperature of 35℃, the weight ratio of 1/10 between CoQ10 and PC, and the weight ratio of 1/3 between cholesterol and PC, the CoQ10 loading reached 8.92%. CoQ10 liposomes were obtained by hydrating CoQ10 proliposomes and the entrapment efficiency of CoQ10 reached 82.28%. The morphology of CoQ10 proliposomes were characterized by SEM, and their solid state was characterized by XRD. The structure of CoQ10 liposomes were characterized by TEM. The particle size distribution of CoQ10 liposomes was determined by DLS. The results indicate that CoQ10 liposomes with particle sizes about 50nm can be easily got from hydrating CoQ10 proliposomes prepared by SEDS.
3. Vitamin D3 (VD_3) proliposomes, consisted of hydrogenated phosphatidycholine (HPC) and VD_3, were prepared using SEDS. The effects of operation conditions (temperature, pressure and components) on the VD_3 loading in VDP were studied. At the optimum conditions of pressure of 8.0MPa, temperature of 45℃, and the weight ratio of 15.0% between VD_3 and HPC, the VD_3 loading reached 12.89%. VD_3 liposomes were obtained by hydrating VD_3 proliposomes and the entrapment efficiency of VD_3 in VD_3 liposomes reached 98.5%. The morphology and structure of proliposomes and liposomes were characterized by SEM, TEM and XRD. The structure of VD_3 nanoparticles in HPC matrix was formed. The size of liposome was determined by Dynamic Light Scattering instrument (DLS). The average diameter of liposomes was about 1μm. The results indicate that VDP can be made by SEDS and liposomes with high entrapment efficiency can be formed easily via the hydration of proliposomes.
4. Proliposomes composed of lutein and hydrogenated phosphatidylcholine were prepared using SEDS. The effects of the process parameters on the lutein loading and the particle sizes of the proliposomes were investigated. HPLC was applied to determine the content of lutein in the samples. At the optimum conditions—temperature of 35℃, pressure of 8MPa and the solution flow rate of 1 ml/min—the lutein loading of the proliposomes reached 55mg/g. The images characterized by SEM were evaluated for the different proliposomes samples in order to study the influences of operational conditions on the particle sizes and morphology. When proliposome was hydrated, the lutein liposome suspensions were formed automatically. The crystallinity of proliposomes was analyzed using DSC to analyze the distribution of lutein in proliposomes. The structure of proliposomes and the lutein liposome was detected by TEM. The results indicate that proliposomes with the high lutein loading was made successfully and the lutein liposome was obtained with the encapsulation efficiency of more than 90% after hydrating proliposomes. The animal experiment results show that there is dose dependence on the lutein liposomes. These results demonstrate that SEDS technique is a simple and effective process for the preparation of proliposomes from which liposome can be easily formed.
引文
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