CN102793669A - 一种ps-脂质体合成工艺 - Google Patents
一种ps-脂质体合成工艺 Download PDFInfo
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Abstract
本发明涉及一种脂质体合成工艺,特别涉及一种PS-脂质体合成工艺,属于生物医药领域,包括以下步骤:(1)、将脂质体、人体非必需氨基酸和三氯甲烷按体积百分比混合均匀;(2)、将(1)中的混合物干馏;(3)、将(2)的混合物真空干燥,然后加入CMF-PBS,使之成悬浊样液体;(4)、将(3)中的悬浊液用超声波粉碎至成透明样液体,然后离心,取上清液;(5)、将(4)中的上清液用SaltoriusFilter(0.22μm)过滤灭菌,制得PS-脂质体溶液。人体非必需氨基酸为磷脂酰丝氨酸和磷脂酰胆碱。本发明的PS-脂质体合成工艺,工艺简单易控制、工艺要求低,且生产过程中无副产物,安全环保。
Description
技术领域
本发明涉及一种脂质体合成工艺,特别涉及一种PS-脂质体合成工艺,属于生物医药领域。
背景技术
21世纪脑研究将是生命科学研究的主流,随着我国人民生活水平的提高和人口的老龄化,对于急性脑血管疾病、神经系统变性病等中枢神经系统疾病新型药物的开发将变为更加迫切。
脂质体(liposome,或称类脂小球,液晶微囊)以磷脂、胆固醇等类脂质为膜材,具有类细胞膜结构,故作为药物的载体,能被单核吞噬细胞系统吞噬,增加药物对淋巴组织的指向性和靶组织的滞留性。近年的生物学研究表明:磷脂酰丝氨酸(简称PS)作为膜表面的标记物在周围组织的巨噬细胞、血管内皮细胞吞噬凋亡细胞的过程中发挥着极为重要的定向诱导作用,巨噬细胞在进行吞噬作用后能够产生转化生长因子-β(Transforming growth factor-β TGF-β)等抗炎性介质,限制炎症反应的发生。因此,本领域技术人员急需研发一种PS-脂质体合成工艺。
发明内容
为解决现在技术中的上述不足,本发明的目的是提供一种PS-脂质体合成工艺,该合成工艺简单易控制、工艺要求低,且生产过程中无副产物。
为实现上述目的,本发明是通过以下技术方案实现的:
一种PS-脂质体合成工艺,包括以下步骤:
(1)、将脂质体、人体非必需氨基酸和三氯甲烷按体积百分比混合均匀;
(2)、将(1)中的混合物干馏;
(3)、将(2)的混合物真空干燥,然后加入CMF-PBS,使之成悬浊样液体;
(4)、将(3)中的悬浊液用超声波粉碎至成透明样液体,然后离心,取上清液;
(5)、将(4)中的上清液用Saltorius Filter(0.22μm)过滤灭菌,制得PS-脂质体溶液。
所述的人体非必需氨基酸为磷脂酰丝氨酸和磷脂酰胆碱。
所述的步骤(1)中的体积百分比为:
脂质体 40%-55%
磷脂酰丝氨酸 30%-48%
磷脂酰胆碱 10%-12%
三氯甲烷 5%-9%。
作为优选的,一种PS-脂质体合成工艺,所述的步骤(1)中的体积百分比为:
脂质体 45%
磷脂酰丝氨酸 38%
磷脂酰胆碱 10%
三氯甲烷 7%。
所述的步骤(4)中的离心温度为4℃。
本发明的PS-脂质体合成工艺,工艺简单易控制、工艺要求低,且生产过程中无副产物,安全环保。
具体实施方式
实施例1
将45ml脂质体、38ml磷脂酰丝氨酸、10ml磷脂酰胆碱与7ml三氯甲烷混合,混合均匀后干馏,将干馏出来的产物真空干燥后加入CMF-PBS成悬浊样液体,然后用超声波粉碎致成透明样溶液,4℃离心分离后取上清液,用Saltorius Filter(0.22μm)过滤灭菌,制得PS-脂质体溶液。
实施例2
将55ml脂质体、30ml磷脂酰丝氨酸、10ml磷脂酰胆碱与5ml三氯甲烷混合,混合均匀后干馏,将干馏出来的产物真空干燥后加入CMF-PBS成悬浊样液体,然后用超声波粉碎致成透明样溶液,4℃离心分离后取上清液,用Saltorius Filter(0.22μm)过滤灭菌,制得PS-脂质体溶液。
实施例3
将40ml脂质体、39ml磷脂酰丝氨酸、12ml磷脂酰胆碱与9ml三氯甲烷混合,混合均匀后干馏,将干馏出来的产物真空干燥后加入CMF-PBS成悬浊样液体,然后用超声波粉碎致成透明样溶液,4℃离心分离后取上清液,用Saltorius Filter(0.22μm)过滤灭菌,制得PS-脂质体溶液。
实施例4
将52ml脂质体、31ml磷脂酰丝氨酸、11ml磷脂酰胆碱与6ml三氯甲烷混合,混合均匀后干馏,将干馏出来的产物真空干燥后加入CMF-PBS成悬浊样液体,然后用超声波粉碎致成透明样溶液,4℃离心分离后取上清液,用Saltorius Filter(0.22μm)过滤灭菌,制得PS-脂质体溶液。
性能测试
将上述实施例1-4制得的PS-脂质体进行粒径、包封率和表面张力的检测。
将PS-脂质体溶液用10mmol/L乙酸铵、50mmol/L Tris溶液(PH=7.5)调配至0.3-0.6g/L,滴在有支持膜的铜网上,滤纸吸干,再滴加钼酸铵,滤纸吸干,自然干燥30min,电镜观察PS-脂质体的结构和粒径大小,在电镜照片上随机取10个不同的视野,分别测量PS-脂质体的直径,将此直径乘以校正系统0.707即为PS-脂质体粒径。
量取PS-脂质体溶液0.5ml,用PBS稀释至5.0ml,4℃下,以37000r/min离心30min。弃上清液以去除包裹的PS,沉积于底部的即为脂质体小囊,将脂质体小囊以PBS稀释至1.0ml。分别取200uL脂质体小囊液和100uL PS-脂质体溶液,加入100g/L Triton 100溶解,用Folin酚试剂法测定其中蛋白质的含量。脂质体小囊液中蛋白质的含量与脂质体溶液中蛋白质的含量的比值即为PS-脂质体的包封率。
取PS-脂质体用表面张力仪测定5min。
测试结果见下表。
表
实施例 | 粒径(um,n=30) | 包封率(%) | 表面张力(mN/m,n=4) |
实施例1 | 0.463 | 60 | 13.25 |
实施例2 | 0.451 | 62 | 13.55 |
实施例3 | 0.461 | 60 | 13.25 |
实施例4 | 0.468 | 65 | 13.80 |
综上所述,本发明的PS-脂质体合成工艺,工艺简单易控制、工艺要求低,且生产过程中无副产物,安全环保。
上述实施例仅用于解释说明本发明的发明构思,而非对本发明权利保护的限定,凡利用此构思对本发明进行非实质性的改动,均应落入本发明的保护范围。
Claims (5)
1.一种PS-脂质体合成工艺,其特征在于:包括以下步骤:
(1)、将脂质体、人体非必需氨基酸和三氯甲烷按体积百分比混合均匀;
(2)、将(1)中的混合物干馏;
(3)、将(2)的混合物真空干燥,然后加入CMF-PBS,使之成悬浊样液体;
(4)、将(3)中的悬浊液用超声波粉碎至成透明样液体,然后离心,取上清液;
(5)、将(4)中的上清液用Saltorius Filter(0.22μm)过滤灭菌,制得PS-脂质体溶液。
2.如权利要求1所述的PS-脂质体合成工艺,其特征在于:所述的人体非必需氨基酸为磷脂酰丝氨酸和磷脂酰胆碱。
3.如权利要求2所述的PS-脂质体合成工艺,其特征在于:所述的步骤(1)中的体积百分比为:
脂质体 40%-55%
磷脂酰丝氨酸 30%-48%
磷脂酰胆碱 10%-12%
三氯甲烷 5%-9%。
4.如权利要求2所述的PS-脂质体合成工艺,其特征在于:所述的步骤(1)中的体积百分比为:
脂质体 45%
磷脂酰丝氨酸 38%
磷脂酰胆碱 10%
三氯甲烷 7%。
5.如权利要求1所述的PS-脂质体合成工艺,其特征在于:所述的步骤(4)中的离心温度为4℃。
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Citations (2)
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CN1731982A (zh) * | 2002-10-25 | 2006-02-08 | 西摩·J·库尔茨 | 治疗胰岛素耐受性、成人发作型糖尿病以及代谢综合症x的方法 |
CN101283987A (zh) * | 2008-05-19 | 2008-10-15 | 浙江大学 | 氯喹脂质体冻干粉针及制备方法 |
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CN1731982A (zh) * | 2002-10-25 | 2006-02-08 | 西摩·J·库尔茨 | 治疗胰岛素耐受性、成人发作型糖尿病以及代谢综合症x的方法 |
CN101283987A (zh) * | 2008-05-19 | 2008-10-15 | 浙江大学 | 氯喹脂质体冻干粉针及制备方法 |
Non-Patent Citations (1)
Title |
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潘卫三主编: "《工业药剂学》", 30 June 2010, article ""第二节被动靶向制剂 2.主动载药技术"", pages: 476-478 * |
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