CN113491775A - 葡萄糖修饰脂质体、载药脂质体及其制备方法和应用 - Google Patents
葡萄糖修饰脂质体、载药脂质体及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及医药技术领域,尤其是涉及一种葡萄糖修饰脂质体、载药脂质体及其制备方法和应用。葡萄糖修饰脂质体,其表面修饰有具有脑靶向作用的葡萄糖配体。载药脂质体,包括所述葡萄糖修饰脂质体和包封于所述脂质体内的药物。本发明的脂质体可将EGCG等药物包埋于其中,减轻外界条件对药物分子结构的影响。并且,本发明的葡萄糖修饰脂质体以及载药脂质体,能抵抗H2O2诱导PC12细胞氧化损伤,并且对人体无毒副作用;具有较强的主动脑靶向性、神经细胞靶向性,可提高血脑屏障穿透率,能够用于制备抗神经细胞氧化损伤的药物。
Description
技术领域
本发明涉及医药技术领域,尤其是涉及一种葡萄糖修饰脂质体、载药脂质体及其制备方法和应用。
背景技术
缺血性脑病是由于脑缺氧或缺血引起脑损伤从而产生的病症,患者常会出现意识改变、肌张力变化等临床病症,严重者将引起诸如脑瘫、癫痫等后遗症。研究表明,脑缺血时供应大脑的氧气和葡萄糖减少,导致生物功能障碍,进而造成氧化应激、过度炎症反应、兴奋性细胞毒性作用、细胞内钙离子超载等,最终导致神经细胞凋亡(Marisol Godínez-Rubí,et al.Oxidative medicine and cellular longevity,2013,3(7):1-16)。抗氧化剂因为强大的抗氧化作用,成为脑损伤治疗的研究热点。表没食子儿茶素没食子酸酯(EGCG,结构式如下I所示)为天然抗氧化剂,可运用于神经保护(Ali Reza Khalatbary,etal.Nutritional Neuroscience,2020,23(4):281-294)。
目前,大量研究表明EGCG具有抗肿瘤、抗炎、抗氧化、抗衰老、抗肥胖、抗紫外、降血压、降血糖、降血脂、预防和治疗心脑血管系统疾病以及调节内分泌、免疫系统等生物活性(张思佳等,中国新药与临床杂志,2013,32(7):521-526)。除此之外,EGCG的神经保护作用也越来越受重视。6-羟基多巴胺是多巴胺能神经毒素,体外实验研究发现EGCG能抵抗6-羟基多巴胺诱导的大鼠PC12细胞和人胶质瘤细胞SH-SY5Y细胞死亡(R.K.Chaturvedi,etal.Neurobiology of Disease,2006,22(2):421-434)。但由于EGCG分子结构的限制,其水溶性好而脂溶性差,将其用于神经保护,血脑屏障穿透率低,且EGCG本身极不稳定,在溶液中易氧化为对人体有害的醌类化合物,且易受环境因素的影响,温度、氧化剂、偏中性或碱性的环境都会严重影响EGCG的稳定性。
血脑屏障(blood brain barrier,BBB)存在于血液循环系统和中枢神经系统之间,主要由内皮细胞、星形胶质细胞、周细胞、基底膜以及内皮细胞之间的紧密连接组成(Patel M M,et al.CNS Drugs,2017,31(2):109-133),且只允许分子量小于400Da,氢键数小于8的小分子亲脂性物质穿过,选择性地将有害或过量的物质排出大脑,维持着大脑内环境的动态平衡(Mignai S,et al.Progress in Ploymer Science,2017,64:23-51)。几乎所有的大分子药物,包括多肽、重组蛋白、单克隆抗体、基于RNA干扰技术的药物以及大部分小分子药物,都不能通过血脑屏障(Reynolds J L,et al.Journal of NeuroimmunePharmacology,2017,12(1):1-5)。纳米载体是递送药物入脑的优良载体,载体较高的比表面积有助于提供较高的药物载量,载体表面电荷有助于对其表面进行修饰,实现靶向特异性,转胞吞是纳米材料穿透血脑屏障的主要方式。载体介导的传递系统(carrier-mediatedtransport system,CMT)是将大脑所需营养物质转运入脑的一种方式,如己糖、Vc、氨基酸、核苷、胺类、多肽与肉碱转运系统等(P.Campos-Bedolla,et al.Archives of MedicalResearch,2014,45(8):610-638)。
因此,为了提高EGCG稳定性和血脑屏障穿透率需对EGCG的结构进行改造或采用药物制剂技术进行剂型设计。
有鉴于此,特提出本发明。
发明内容
本发明的第一目的在于提供一种葡萄糖修饰脂质体,以解决现有技术中存在的药物血脑屏障穿透率低等技术问题。
本发明的第二目的在于提供一种载药脂质体,解决了单独药物稳定性差和血脑屏障穿透率低等技术问题。
本发明的第三目的在于提供载药脂质体的制备方法。
本发明的第四目的在于提供葡萄糖修饰脂质体或载药脂质体在制备抗神经细胞氧化损伤的药物中的应用。
为了实现本发明的上述目的,特采用以下技术方案:
葡萄糖修饰脂质体,其表面修饰有具有脑靶向作用的葡萄糖配体。
在本发明的具体实施方式中,所述葡萄糖配体的结构式如下:
在本发明的具体实施方式中,所述聚乙二醇结构的重复单元数n=1~100。
在本发明的具体实施方式中,所述脂质体的膜材包括磷脂、胆固醇和所述葡萄糖配体。
本发明还提供了载药脂质体,包括上述任意一种所述葡萄糖修饰脂质体和包封于所述脂质体内的药物。
在本发明的具体实施方式中,所述药物为水溶性药物,所述水溶性药物包括但不仅限于EGCG、L-抗坏血酸、阿霉素、阿糖胞苷和黄连素及各自的药学上可接受的盐或水合物中的至少一种。
本发明的脂质体可将水溶性药物如EGCG包埋于其中,解决了EGCG等水溶性药物自身不稳定的问题。钠离子非依赖型的葡萄糖转运蛋白1(GLUT1)在脑毛细血管内皮细胞表面过表达,在血脑屏障的众多转运系统中,GLUT1被认为是最受欢迎的CMT转运系统之一,与众多其他位于BCECs中的转运体相比,其表达水平要高得多。BBB上过表达的GLUT1可以转运葡萄糖进脑,以供脑内的葡萄糖消耗。本发明将葡萄糖配体修饰于脂质体表面,可通过GLUT1介导的内吞透过血脑屏障,实现主动脑靶向,并且能抵抗,使得到的载药脂质体具有较好的血脑屏障穿透率。
在本发明的具体实施方式中,所述载药脂质体的包封率≥70%。
本发明还提供了上述任意一种所述载药脂质体的制备方法,采用薄膜水化法制备所述载药脂质体。
在本发明的具体实施方式中,所述制备方法包括如下步骤:
以药物作为活性成分,以磷脂、胆固醇和葡萄糖配体作为膜材,采用薄膜水化法制备所述载药脂质体。
对于载药脂质体,通常易于包覆脂溶性药物,且具有良好的包封率;但水溶性药物难于包裹在脂质体的内水相,存在内水相装载药物太少,或装载药物易泄露等问题。本发明通过采用薄膜水化法,并通过对各组分用量进行调控,能够兼顾保证药物包封率以及包封稳定性等。
在本发明的具体实施方式中,所述薄膜水化法包括:脂质膜于PBS缓冲液中震摇处理,然后进行超声处理,再进行离心处理,将得到的上清液进行超滤处理,除去未包封的活性成分,收集截留液进行离心处理。
在本发明的具体实施方式中,采用旋转蒸发的方式将含所述膜材和所述活性成分的有机溶液蒸干得到所述脂质膜。进一步的,采用减压旋转蒸发的方式,温度为35~40℃。
在本发明的具体实施方式中,所述磷脂与所述胆固醇的摩尔比为1~10﹕1~2,所述葡萄糖配体的摩尔量与所述磷脂和所述胆固醇的总摩尔量的比例为1﹕(4~100)。
在本发明的具体实施方式中,按重量百分数计算,所述活性成分占所述磷脂和所述胆固醇总重量的0.1%~50%。
在本发明的具体实施方式中,所述有机溶液中,所述磷脂、所述胆固醇和所述葡萄糖配体的摩尔比为(12.9~13.1)﹕(5.9~6.1)﹕1,如13﹕6﹕1。
在实际操作中,根据磷脂的摩尔分子量计算磷脂的用量。
在本发明的具体实施方式中,所述有机溶液中,所述活性成分与所述膜材的质量比为1﹕(21.2~22.8),如1﹕22。
在本发明的具体实施方式中,所述有机溶液中的有机溶剂包括氯仿和甲醇。进一步的,所述氯仿和甲醇的体积比为(1.9~2.1)﹕1,如2﹕1。
在本发明的具体实施方式中,所述活性成分与所述有机溶剂的用量比为1mg﹕(0.8~1.2)mL。
在本发明的具体实施方式中,所述PBS缓冲液与所述有机溶剂的体积比为1﹕(0.8~1.2),如1﹕1。进一步的,所述PBS缓冲液的pH为7.4。
本发明还提供了上述任意一种所述葡萄糖修饰脂质体或任意一种所述载药脂质体在制备抗神经细胞氧化损伤的药物中的应用。
与现有技术相比,本发明的有益效果为:
(1)本发明的葡萄糖修饰脂质体可将药物包埋于其中,减轻外界条件对药物分子结构的影响;
(2)本发明的葡萄糖修饰脂质体以及载药脂质体,能抵抗H2O2诱导PC12细胞氧化损伤,并且对人体无毒副作用;具有较强的主动脑靶向性、神经细胞靶向性,可提高血脑屏障穿透率,能够用于制备抗神经细胞氧化损伤的药物。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例1制得的载药脂质体EGCG-glu-Lip的粒径分布图;
图2为本发明实施例1制得的载药脂质体EGCG-glu-Lip的电位分布图;
图3为本发明的色谱条件下空白溶剂、EGCG、Lip、EGCG-Lip对应的色谱图;
图4为本发明实施例2制得的葡萄糖修饰脂质体glu-Lip和比较例1制得的脂质体Lip在bEnd.3细胞和PC12细胞上摄取的流式数据(n=3,mean±SD;**P<0.01);
图5为本发明实施例2制得的葡萄糖修饰脂质体glu-Lip和比较例1制得的脂质体Lip在bEnd.3细胞和PC12细胞上摄取的共聚焦图;
图6为本发明实施例1制得的载药脂质体EGCG-glu-Lip、比较例2制得的EGCG脂质体EGCG-Lip和EGCG在PC12细胞和bEnd.3细胞上的细胞毒性;
图7为本发明实施例1制得的载药脂质体EGCG-glu-Lip、比较例2制得的EGCG脂质体EGCG-Lip和EGCG抵抗H2O2诱导PC12细胞损伤的MTT图(n=3,mean±SD;*P<0.05,**P<0.01,***P<0.001versus H2O2);
图8为本发明实施例1制得的载药脂质体EGCG-glu-Lip、比较例2制得的EGCG脂质体EGCG-Lip和EGCG抵抗H2O2诱导PC12细胞损伤的ROS流式图(n=3,mean±SD;****P<0.0001);
图9为本发明实施例1制得的载药脂质体EGCG-glu-Lip、比较例2制得的EGCG脂质体EGCG-Lip和EGCG抵抗H2O2诱导PC12细胞损伤的ROS共聚焦图。
具体实施方式
下面将结合附图和具体实施方式对本发明的技术方案进行清楚、完整地描述,但是本领域技术人员将会理解,下列所描述的实施例是本发明一部分实施例,而不是全部的实施例,仅用于说明本发明,而不应视为限制本发明的范围。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
葡萄糖修饰脂质体,其表面修饰有具有脑靶向作用的葡萄糖配体。
在本发明的具体实施方式中,所述葡萄糖配体的结构式如下:
在本发明的具体实施方式中,所述聚乙二醇结构的重复单元数n=1~100。
针对不同分子量的葡萄糖配体,其结构式可以如下:
在本发明的具体实施方式中,所述脂质体的膜材包括磷脂、胆固醇和所述葡萄糖配体。
脂质体是由两亲性分子在水相中分散开时,自然形成封闭型的囊泡,包括内部水相和双分子层脂相,胆固醇镶嵌在脂质体脂相间。上述葡萄糖配体是由靶向分子葡萄糖,中间的亲水性Linker和亲脂性分子胆固醇构成,在制备脂质体的过程中,首先形成脂质膜,水化过程中,脂质膜在水中分散会自然形成封闭型囊泡,由于胆固醇是镶嵌在脂相中,但中间的Linker以及靶向分子本身的亲水性,会让靶向分子向外延伸使其暴露在水相中而不是包埋在脂相中,从而使靶向分子暴露在脂质体表面。
在本发明的具体实施方式中,所述磷脂包括大豆磷脂、卵磷脂、磷脂酰乙醇胺、磷酯酰丝氨酸、磷脂酰肌醇、磷脂酰甘油和二磷脂酰甘油的至少一种,优选为卵磷脂或大豆磷脂。
本发明还提供了载药脂质体,包括上述任意一种所述葡萄糖修饰脂质体和包封于所述脂质体内的药物。
在本发明的具体实施方式中,所述药物为水溶性药物。
在本发明的具体实施方式中,所述水溶性药物包括EGCG、L-抗坏血酸、阿霉素、阿糖胞苷和黄连素及各自的药学上可接受的盐或水合物中的至少一种。进一步的,所述药物为EGCG或其药学上可接受的盐或水合物。
本发明的脂质体可将水溶性药物如EGCG包埋于其中,解决了EGCG等水溶性药物自身不稳定的问题。钠离子非依赖型的葡萄糖转运蛋白1(GLUT1)在脑毛细血管内皮细胞表面过表达,在血脑屏障的众多转运系统中,GLUT1被认为是最受欢迎的CMT转运系统之一,与众多其他位于BCECs中的转运体相比,其表达水平要高得多。BBB上过表达的GLUT1可以转运葡萄糖进脑,以供脑内的葡萄糖消耗。本发明将葡萄糖配体修饰于脂质体表面,可通过GLUT1介导的内吞透过血脑屏障,实现主动脑靶向,并且能抵抗H2O2诱导PC12细胞氧化损伤,使得到的载药脂质体具有较好的血脑屏障穿透率。
在本发明的具体实施方式中,所述载药脂质体具有核壳结构,所述核包括EGCG或其药学上可接受的盐或水合物;所述壳包括磷脂、胆固醇和所述葡萄糖配体。
在本发明的具体实施方式中,所述载药脂质体的包封率≥70%。
本发明还提供了上述任意一种所述载药脂质体的制备方法,采用薄膜水化法制备所述载药脂质体。
在本发明的具体实施方式中,所述制备方法包括如下步骤:
以EGCG作为活性成分,以磷脂、胆固醇和葡萄糖配体作为膜材,采用薄膜水化法制备所述载药脂质体。
在本发明的具体实施方式中,所述薄膜水化法包括:脂质膜于PBS缓冲液中震摇处理,然后进行超声处理,再进行离心处理,将得到的上清液进行超滤处理,除去未包封的活性成分,收集截留液进行离心处理。
在上述薄膜水化法中,震摇处理后得到不均匀的脂质体,超声处理以除去多余的脂质膜材,超滤处理除去溶于PBS缓冲液的活性成分如EGCG,最终得到粒径均匀的载药脂质体。
在本发明的具体实施方式中,采用旋转蒸发的方式将含所述膜材和所述活性成分的有机溶液蒸干得到所述脂质膜。进一步的,采用减压旋转蒸发的方式,温度为35~40℃。在实际操作中,采用常规的减压旋转蒸发仪即可。
在本发明的具体实施方式中,所述磷脂与所述胆固醇的摩尔比为1~10﹕1~2,所述葡萄糖配体的摩尔量与所述磷脂和所述胆固醇的总摩尔量的比例为1﹕(4~100)。
在本发明的具体实施方式中,按重量百分数计算,所述活性成分占所述磷脂和所述胆固醇总重量的0.1%~50%。
在本发明的具体实施方式中,所述有机溶液中,所述磷脂、所述胆固醇和所述葡萄糖配体的摩尔比为(12.9~13.1)﹕(5.9~6.1)﹕1,如13﹕6﹕1。
在本发明的具体实施方式中,所述有机溶液中,所述活性成分与所述膜材的质量比为1﹕(21.2~22.8),如1﹕21.2、1﹕21.4、1﹕21.6、1﹕21.8、1﹕22、1﹕22.2、1﹕22.4、1﹕22.6、1﹕22.8等等。
在本发明的具体实施方式中,所述有机溶液中的有机溶剂包括氯仿和甲醇。进一步的,所述氯仿和甲醇的体积比为(1.9~2.1)﹕1,如2﹕1。
在本发明的具体实施方式中,所述活性成分与所述有机溶剂的用量比为1mg﹕(0.8~1.2)mL。
在本发明的具体实施方式中,所述PBS缓冲液与所述有机溶剂的体积比为1﹕(0.8~1.2),如1﹕1。进一步的,所述PBS缓冲液的pH为7.4。
在本发明的具体实施方式中,所述震摇处理的条件包括:温度为35~40℃,转速150~200rpm,时间为30~40min。
在本发明的具体实施方式中,所述超声处理频率为60~100W,所述超声处理为间歇超声,所述间歇超声的时间为5~8min。进一步的,在冰浴条件下进行所述超声处理。进一步的,所述间歇超声可以为工作5s,停止5s。
在本发明的具体实施方式中,第一次所述离心处理的转速为8000~12000rpm,优选为10000rpm;第一次所述离心处理的时间为5~15min。
在本发明的具体实施方式中,采用截留分子量为2KD~3KD的超滤设备进行所述超滤处理。进一步的,所述超滤设备可以为超滤管,但不局限于此。
在本发明的具体实施方式中,将所述截留液进行离心处理的条件包括:离心转速为12000~16000rpm,离心时间为30~40min。进一步的,所述离心转速可以为14000rpm,离心时间可以为30min
本发明还提供了上述任意一种所述葡萄糖修饰脂质体或任意一种所述载药脂质体在制备抗神经细胞氧化损伤的药物中的应用。
下述实施例中采用的葡萄糖配体结构如下,但不局限于此,Linker对应不同分子量的聚乙二醇的制备方法相同,区别仅在于聚乙二醇种类的替换:
实施例1
本实施例提供了载药脂质体EGCG-glu-Lip及其制备方法,所述制备方法包括如下步骤:
(1)精密称取大豆磷脂(SPC)17.45mg、胆固醇(chol)3.89mg、葡萄糖配体1.31mg、EGCG 1mg置于50mL茄形瓶中,加入1mL体积比为2﹕1的氯仿和甲醇,混合均匀后置于37℃的减压旋转蒸发仪上蒸干成脂质膜,抽干有机溶剂。
(2)向步骤(1)得到的脂质膜中加入1mL pH为7.4的PBS缓冲液,置于37℃、180rpm摇床震摇30min,水化完成后将吸出脂质体置于1.5mL的EP管中,于冰浴条件下,80W间歇超声6min,然后于10000rpm转速离心10min,取上清液置于3KD超滤管中,收集截留液于14000rpm转速离心30min,得到乳状载药脂质体EGCG-glu-Lip。
实施例2
本实施例提供了葡萄糖修饰脂质体glu-Lip及其制备方法,所述制备方法参考实施例1,区别仅在于:在步骤(1)中不加入EGCG,其余操作相同。
实施例3
本实施例提供了不同药脂比、不同大豆磷脂(SPC)与胆固醇(chol)比例对应制备得到的载药脂质体EGCG-Lip,各载药脂质体的制备方法参考实施例1,区别在于,不添加葡萄糖配体,并对各组分用量进行调整。各载药脂质体EGCG-Lip的药脂比、大豆磷脂(SPC)与胆固醇(chol)的摩尔比例见表1。其中,药脂比是指:EGCG与大豆磷脂和胆固醇的总和的质量比。
表1不同载药脂质体EGCG-Lip的用量信息
比较例1
比较例1提供了空白脂质体Lip及其制备方法,其制备方法参考实施例1,区别在于:
不添加EGCG,不添加葡萄糖配体,并调整大豆磷脂和胆固醇的摩尔比为65﹕35,称取大豆磷脂17.45mg、胆固醇4.54mg,其余操作与实施例1一致。
比较例2
比较例2提供了EGCG脂质体EGCG-Lip及其制备方法,其制备方法参考实施例1,区别在于:
不添加葡萄糖配体,并调整大豆磷脂和胆固醇的摩尔比为65﹕35,称取大豆磷脂17.45mg、胆固醇4.54mg、EGCG 1mg,其余操作与实施例1一致。
实验例1
将实施例1制备的载药脂质体EGCG-glu-Lip用纯水稀释10倍,置于脂质体样品皿中,进行动态光散射分析仪测定脂质体的粒径大小与分布,测定结果如图1所示,EGCG-glu-Lip粒径为158.7nm,分散系数PDI为0.256,表明载药脂质体粒径均匀。
取上述稀释处理的载药脂质体EGCG-glu-Lip样品,置于测定电位的样品池中,进行电位测定,测定结果如图2所示,电位值为2.73mV,表明载药脂质体EGCG-glu-Lip具有良好的带电性质,进入体内后不至于被肾脏快速代谢也不至于被网状内皮系统快速清除。
实验例2
载药脂质体EGCG-glu-Lip包封率的测定
采用下述HPLC条件对EGCG含量进行检测:
色谱柱:Gemini 5μm C18 110A(150×4.6mm),柱温:30℃,流动相:0.1%磷酸水溶液-甲醇(v﹕v=70﹕30),流速:1.0mL/min,检测波长:279nm,进样量:20μL,供试品所用溶剂:流动相。
在上述色谱条件下,单独的EGCG在5.254min出峰,峰型良好,EGCG峰如图3中(B)所示。图3中(A)为上述色谱条件下,单独溶剂(流动相)的色谱图,在EGCG出峰时间没有其他峰,表明溶剂不影响EGCG峰型。图3中(C)为比较例1的空白脂质体Lip在甲醇涡旋破碎后的色谱图,在5min左右没有出峰,表明脂质材料在此色谱条件下对EGCG峰没有影响。图3中(D)为实施例1制得的脂质体EGCG-glu-Lip在甲醇涡旋破碎后的色谱图,其中对应的EGCG峰的峰型良好,表明此色谱条件可以测定载EGCG的脂质体的EGCG包封含量。
采用HPLC法测定脂质体包封率,采用上述HPLC条件进行含量测定。配制EGCG浓度为2.5μg/L~100μg/L之间的一系列标准溶液(溶剂为流动相),进样测试色谱图,绘制EGCG浓度(x)与EGCG峰面积(y)的关系曲线,为y=19.781x-4.2714,R2=0.9999。将实施例1制备的载药脂质体EGCG-glu-Lip用10倍体积甲醇稀释后涡旋6min,使脂质膜破碎,然后于10000rpm离心10min,吸取上清用流动相对半稀释进样,得出峰面积后根据标准曲线,得到进样样品中EGCG浓度,进而计算载药脂质体EGCG-glu-Lip的EGCG的包封率。测定结果表明,载药脂质体EGCG-glu-Lip的包封率可达73.33%。
按照上述方法测算实施例3制备得到的不同载药脂质体EGCG-Lip的包封率,结果如下表2所示。
表2不同载药脂质体EGCG-Lip的包封率
实验例3
葡萄糖修饰脂质体glu-Lip的脑靶向性定量测定
采用bEnd.3细胞(小鼠脑微血管内皮细胞)作为血脑屏障模型,考察脂质体在bEnd.3细胞上的摄取。操作步骤如下:
将bEnd.3细胞复苏后,用10%胎牛血清、1%双抗的DMEM培养基,37℃、5%CO2的环境下培养两周,每隔一天换液一次,待细胞长满后进行细胞传代操作。取对数生长期的细胞按3×105接种于12孔板内,培养24h,培养结束后于避光条件下分别加入含有脂性荧光物质CFPE的空白脂质体Lip以及葡萄糖修饰脂质体glu-Lip,设3个复孔,保证CFPE在培养液中的浓度为3μmol/mL,继续孵育2.5h。孵育结束后,弃去培养基,PBS缓冲液(pH 7.4)清洗三遍,胰酶消化,离心,再用PBS缓冲液(pH 7.4)清洗一遍,350μL的PBS缓冲液(pH 7.4)重悬细胞,置于流式细胞仪上,选择FITC通道,测定细胞平均荧光值。结果如图4中(A)所示,glu-Lip处理组的荧光强度是Lip处理组的荧光强度的1.5倍,结果具有显著性差异,表明葡萄糖修饰脂质体glu-Lip可增强血脑屏障透过率。
葡萄糖修饰脂质体glu-Lip的脑靶向性定性评价
取对数生长期的bEnd.3细胞按5×103接种于共聚焦皿内,培养24h,培养结束后于避光条件下分别加入含有脂性荧光物质CFPE的空白脂质体Lip和葡萄糖修饰脂质体glu-Lip,设3个复孔,保证CFPE在培养液中的浓度为3μmol/mL,继续孵育2.5h。孵育结束后,弃去培养基,PBS缓冲液(pH7.4)清洗三遍,4%多聚甲醛固定30min,PBS缓冲液(pH 7.4)清洗三遍,0.1mg/mL的DAPI染色细胞核5min,PBS缓冲液(pH 7.4)清洗三遍,置于激光共聚焦显微镜60X油倍镜下,观察Amlyanl和DAPI通道下的荧光强度。结果如图5所示,绿色荧光表示脂质体穿透细胞膜进入细胞的量,在bEnd.3细胞内的绿色荧光强度glu-Lip处理组明显强于Lip处理组,进一步说明葡萄糖修饰脂质体glu-Lip可增强血脑屏障透过率。
实验例4
葡萄糖修饰脂质体glu-Lip的神经细胞靶向性定量测定
采用PC12细胞(大鼠肾上腺嗜铬细胞瘤细胞)作为神经细胞模型,考察脂质体在PC12细胞上的摄取。操作步骤如下:
将PC12细胞复苏后,用10%胎牛血清、1%双抗的DMEM培养基,37℃、5%CO2的环境下培养两周,每隔一天换液一次,待细胞长满后进行细胞传代操作。取对数生长期的PC12细胞按3×105接种于12孔板内,培养24h,培养结束后于避光条件下分别加入含有脂性荧光物质CFPE的空白脂质体Lip以及葡萄糖修饰脂质体glu-Lip,设3个复孔,保证CFPE在培养液中的浓度为3μmol/mL,继续孵育2.5h。孵育结束后,弃去培养基,PBS缓冲液(pH 7.4)清洗三遍,胰酶消化,离心,再用PBS缓冲液(pH 7.4)清洗一遍,350uL的PBS缓冲液(pH 7.4)重悬细胞,置于流式细胞仪上,选择FITC通道,测定细胞平均荧光值。结果如图4中(B)所示,glu-Lip处理组的荧光强度是Lip处理组的荧光强度的1.3倍,结果具有显著性差异,表明葡萄糖修饰脂质体glu-Lip可增强神经细胞靶向性。
葡萄糖修饰脂质体glu-Lip的神经细胞靶向性定性评价
取对数生长期的PC12细胞按5×103接种于共聚焦皿内,培养24h,培养结束后于避光条件下分别加入含有脂性荧光物质CFPE的空白脂质体Lip以及葡萄糖修饰脂质体glu-Lip,设3个复孔,保证CFPE在培养液中的浓度为3μmol/mL,继续孵育2.5h。孵育结束后,弃去培养基,PBS缓冲液(pH7.4)清洗三遍,4%多聚甲醛固定30min,PBS缓冲液(pH 7.4)清洗三遍,0.1mg/mL的DAPI染色细胞核5min,PBS缓冲液(pH 7.4)清洗三遍,置于激光共聚焦显微镜60X油倍镜下,观察Amlyanl和DAPI通道下的荧光强度。结果如图5所示,绿色荧光表示脂质体穿透细胞膜进入细胞的量,在PC12细胞内的绿色荧光强度glu-Lip处理组明显强于Lip处理组,进一步说明葡萄糖修饰脂质体glu-Lip具有神经细胞靶向性。
实验例5
载药脂质体EGCG-glu-Lip的细胞毒性考察
根据包封率计算EGCG-Lip,EGCG-glu-Lip中EGCG浓度,用培养基将EGCG-Lip,EGCG-glu-Lip,EGCG分别逐步稀释为2.5μmol/mL、5μmol/mL、10μmol/mL、20μmol/mL、40μmol/mL、60μmol/mL、80μmol/mL、160μmol/mL备用。取对数生长期的bEnd.3细胞和PC12细胞按2×103接种于96孔板内,培养24h后,分别加入上述系列浓度的载EGCG脂质体EGCG-Lip、EGCG-glu-Lip或EGCG溶液,设6个复孔,继续孵育24h。孵育结束后,弃去培养基,向细胞孔内加入200μL的0.5mg/mL的MTT溶液,孵育4h后,小心吸去上层培养基,加入150μL DMSO,置于酶标仪中震荡180s,测定490nm波长处的吸光值A测定。以DMSO的吸光度值A空白作为空白,以未加药处理的细胞孔同法操作测得的吸光度值A对照作为对照,计算各孔细胞的存活率,存活率(%)=(A测定-A空白)/(A对照-A空白)×100%。结果如图6所示,所有脂质体在EGCG 2.5~160μmol/L浓度下,对bEnd.3细胞均未见明显毒性,对PC12细胞在EGCG 2.5~80μmol/L未见明显毒性,基于此可选用EGCG 2.5~80μmol/L浓度范围考察其神经保护作用。
实验例6
载药脂质体EGCG-glu-Lip的抵抗神经细胞氧化损伤能力考察
采用H2O2氧化损伤PC12细胞作为神经细胞损伤模型。具体步骤如下:
根据包封率计算EGCG-Lip,EGCG-glu-Lip中EGCG浓度,用培养基将EGCG-Lip,EGCG-glu-Lip,EGCG分别逐步稀释为0μmol/mL、5μmol/mL、10μmol/mL、20μmol/mL、40μmol/mL、60μmol/mL、80μmol/mL备用。取对数生长期的PC12细胞按1×104接种于96孔板内,培养24h,分别加入上述系列浓度的载EGCG脂质体EGCG-Lip、EGCG-glu-Lip或EGCG溶液,设6个复孔,设置空白对照和阴性对照,继续孵育保护2.5h。孵育结束后弃去培养基,PBS缓冲液(pH7.4)清洗一遍,加入800μmoL/L的H2O2,氧化损伤2h,损伤结束后,弃去培养基,PBS缓冲液(pH7.4)清洗一遍,向细胞孔内加入200μL 0.5mg/mL的MTT溶液,孵育4h后,小心吸去上层培养基,加入150μL DMSO,置于酶标仪中震荡180s,测定570nm波长处的吸光值A测定。以DMSO的吸光度值A空白作为空白,以未加药且未H2O2损伤的细胞孔同法操作测得的吸光度值A对照作为对照,计算各孔细胞的存活率,存活率(%)=(A测定-A空白)/(A对照-A空白)×100%。结果如图7中(A)、(B)、(C)所示,EGCG在5~80μmol/L浓度之间都能抵抗氧化损伤,在5~40μmol/L浓度范围内随浓度升高抵抗氧化损伤能力增强,选用40μmol/L浓度下,EGCG-Lip,EGCG-glu-Lip,EGCG抵抗氧化损伤能力,结果如图7中的(D)所示,EGCG-glu-Lip的神经保护能力是EGCG-Lip的1.19倍,是EGCG溶液的1.43倍,结果具有显著性差异,抵抗氧化损伤能力排序为EGCG-glu-Lip>EGCG-Lip>EGCG,表明将EGCG包封于脂质体内可以显著增强EGCG的神经保护作用,葡萄糖修饰后能进一步增强EGCG的神经保护作用。
实验例7
载药脂质体EGCG-glu-Lip抵抗神经细胞氧化损伤的ROS指标定量评价
取对数生长期的PC12细胞按6×105接种于6孔板内,培养24h,分别加入40μmol/L的EGCG浓度的EGCG-Lip,EGCG-glu-Lip,EGCG,设3个复孔,设置空白对照和阴性对照,继续孵育保护2.5h。孵育结束后弃去培养基,PBS缓冲液(pH 7.4)清洗一遍,除空白对照组外,每孔加入800μmoL/L的H2O2,氧化损伤2h,损伤结束后,弃去培养基,PBS缓冲液(pH 7.4)清洗一遍,装载DCFH-DA探针,将探针以1﹕1000用培养液稀释,染色30min。弃去含探针培养液,PBS缓冲液(pH 7.4)清洗三遍,胰酶消化,含血清培养液终止消化,吹打细胞,收集离心,1mLPBS缓冲液(pH 7.4)重悬离心,400μL PBS缓冲液(pH 7.4)重悬细胞,置于流式细胞仪上,选择FITC通道,测定细胞平均荧光值。结果如图8所示,不同EGCG脂质体或EGCG溶液处理细胞后,细胞内ROS含量比较:EGCG-glu-lip<EGCG-Lip<EGCG,且EGCG脂质体处理后的细胞内ROS含量显著低于EGCG溶液处理后细胞内的ROS含量。表明,将EGCG包封于脂质体内可以通过显著降低细胞内活性氧含量抵抗神经细胞氧化应激损伤,葡萄糖修饰的EGCG脂质体能进一步增强EGCG抵抗氧化应激的能力。
载药脂质体EGCG-glu-Lip抵抗神经细胞氧化损伤的ROS指标定性评价
取对数生长期的PC12细胞按5×103接种于共聚焦皿内,培养24h,分别加入40μmol/L的EGCG浓度的EGCG-Lip,EGCG-glu-Lip,EGCG,设3个复孔,设置空白对照和阴性对照,继续孵育2.5h。孵育结束后弃去培养基,PBS缓冲液(pH 7.4)清洗一遍,除空白对照组外,每孔加入800μmoL/L的H2O2氧化损伤2h,损伤结束后,弃去培养基,PBS缓冲液(pH 7.4)清洗一遍,装载DCFH-DA探针,将探针以1﹕1000用培养液稀释,染色30min。弃去含探针培养液,PBS缓冲液(pH 7.4)清洗三遍,4%多聚甲醛固定细胞30min,PBS缓冲液(pH 7.4)清洗三遍,置于激光共聚焦显微镜20X倍镜下,观察DCF通道下的荧光强度。结果如图9所示,红色荧光表示细胞内ROS含量,只经H2O2处理后的PC12细胞内ROS显著升高,经EGCG处理后,ROS含量降低,证明EGCG抗氧化能力,经不同EGCG脂质体或EGCG溶液处理细胞后,细胞内ROS含量定性比较:EGCG-glu-lip<EGCG-Lip<EGCG,进一步证明,将EGCG包封于脂质体内可以显著降低细胞内活性氧含量,从而抵抗氧化应激,葡萄糖修饰的EGCG脂质体能进一步增强EGCG抵抗氧化应激能力。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (10)
1.葡萄糖修饰脂质体,其特征在于,其表面修饰有具有脑靶向作用的葡萄糖配体。
3.根据权利要求1所述的葡萄糖修饰脂质体,其特征在于,所述脂质体的膜材包括磷脂、胆固醇和所述葡萄糖配体;
优选的,所述磷脂包括大豆磷脂、卵磷脂、磷脂酰乙醇胺、磷酯酰丝氨酸、磷脂酰肌醇、磷脂酰甘油和二磷脂酰甘油的至少一种;
优选的,所述磷脂为卵磷脂或大豆磷脂。
4.载药脂质体,其特征在于,包括权利要求1-3任一项所述的葡萄糖修饰脂质体和包封于所述葡萄糖修饰脂质体内的药物。
5.根据权利要求4所述的载药脂质体,其特征在于,所述药物为水溶性药物;
优选的,所述水溶性药物包括EGCG、L-抗坏血酸、阿霉素、阿糖胞苷和黄连素及各自的药学上可接受的盐或水合物中的至少一种。
6.根据权利要求4所述的载药脂质体,其特征在于,所述载药脂质体的包封率≥70%。
7.权利要求4-6任一项所述的载药脂质体的制备方法,其特征在于,采用薄膜水化法制备所述载药脂质体。
8.根据权利要求7所述的载药脂质体的制备方法,其特征在于,包括如下步骤:
以药物作为活性成分,以磷脂、胆固醇和葡萄糖配体作为膜材,采用薄膜水化法制备所述载药脂质体;
所述薄膜水化法包括:脂质膜于PBS缓冲液中震摇处理后,进行超声处理,再进行离心处理,将得到的上清液进行超滤处理,除去未包封的活性成分,收集截留液进行离心处理;
优选的,采用旋转蒸发的方式将含所述膜材和所述活性成分的有机溶液蒸干得到所述脂质膜。
9.根据权利要求8所述的载药脂质体的制备方法,其特征在于,所述磷脂与所述胆固醇的摩尔比为(1~10)﹕(1~2),所述葡萄糖配体的摩尔量与所述磷脂和所述胆固醇的总摩尔量的比例为1﹕(4~100);
优选的,按重量百分数计算,所述活性成分占所述磷脂和所述胆固醇的总重量的0.1%~50%;
优选的,所述磷脂与所述胆固醇的摩尔比为(12.9~13.1)﹕(5.9~6.1);
优选的,所述葡萄糖配体的摩尔量与所述磷脂和所述胆固醇的总摩尔量的比例为1﹕(18.8~19.2);
优选的,所述活性成分与所述膜材的质量比为1﹕(21.2~22.8)。
10.权利要求1-3任一项所述的葡萄糖修饰脂质体或权利要求4-6任一项所述的载药脂质体在制备抗神经细胞氧化损伤的药物中的应用。
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