CN111588703A - 一种超分子细胞载体、载药体系及其制备方法 - Google Patents
一种超分子细胞载体、载药体系及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种超分子细胞载体、载药体系及其制备方法,涉及超分子化学,超分子材料和细胞制剂技术领域。基于超分子主客体作用构建的超分子细胞载体可实现基于细胞功能的靶向递送效果,具有高生物相容性、高生理屏障透过性和高靶向性。不需要对细胞表面进行共价键修饰,对修饰的细胞生理功能没有影响。本发明提供的超分子细胞载体的制备方法具有制备工艺简单、快速、条件温和和普适性的优势,该方法具有生物正交性。此外,还提供了一种载药体系,可以实现靶向药物治疗的载药。
Description
技术领域
本发明涉及超分子化学,超分子材料和细胞制剂技术领域,具体而言,涉及一种超分子细胞载体、载药体系及其制备方法。
背景技术
炎症与人体各种疾病息息相关,其中也包括癌症、神经系统疾病等重大疾病。然而目前传统药物制剂以及人工合成的靶向制剂对这些重大疾病的治疗效果均不明显,可能与血液循环过程中网状内皮系统的清除作用、到达病变组织前的生理屏障作用以及对病变组织较弱的靶向选择性等因素有关,这些影响因素都会影响药物的最终作用浓度和治疗效果。因此,针对这些与炎症相关的重大疾病,寻找新的递送载体和新的靶向作用途径,开发出新一代具有高生物相容性、高生理屏障透过性、高靶向性的药物制剂是研究和临床上亟待解决的问题。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种超分子细胞载体、载药体系及其制备方法以解决上述技术问题。
本发明是这样实现的:
一种超分子细胞载体,其包括通过主客体作用相互连接的第一部分和第二部分,第一部分为大环主体分子修饰的第一细胞,第二部分为客体分子修饰的纳米粒子或客体分子修饰的第二细胞,第一部分中的大环主体分子通过偶联嵌膜材料嵌入第一细胞的细胞膜中。
细胞是构成生物体结构和进行生物功能的基本单位,若将细胞作为药物递送载体则具有许多天然的优势,但是目前关于利用细胞作为药物载体的相关研究较少,且细胞载体的构建方法也存在一些不可避免的缺陷。其中一种是通过细胞对药物载体的内吞作用来实现细胞对药物载体的负载,然而被吞噬的药物载体可能在胞内环境中发生降解并引起细胞毒性,从而影响基于细胞生理功能的药物递送。还有一种是通过细胞表面与药物载体的共价结合或者特定的配体-受体结合来实现细胞对药物载体的负载,其中共价结合方式涉及到在细胞膜上进行复杂的多步化学反应,可能会影响细胞活性;而通过特定配体-受体相互作用的结合方式仅限于表达相关受体的特定细胞,其应用范围局限性较大。
发明人创造性的提供了一种嵌膜材料嵌入第一细胞的细胞膜的思路,避免了细胞表面与药物载体的共价结合导致细胞活性下降,同时也不仅限于表达相关受体的特定细胞,本发明提供的第一细胞可以根据载药需求进行自适应调整,其应用范围广。在嵌膜材料的一端偶联大环主体分子,大环主体分子可以通过主客体作用与客体分子连接,从而组成超分子细胞载体。
嵌膜材料与细胞膜的结构成分及其类似,通过疏水作用力与细胞膜表面的磷脂层自组装结合。
主客体化学是近些年来兴起的新的研究方向,它是通过主体分子与客体分子之间以非共价键的方式产生键合,研究较多的是β-环糊精与金刚烷的主客体作用,在水中客体分子金刚烷由于疏水性会自动与主体分子环糊精的疏水性空腔结合,形成较为稳定的主客体作用产物。
通过主客体作用以及大环主体分子的偶联嵌膜材料嵌入第一细胞构建的超分子细胞载体可以实现基于细胞功能的靶向递送效果。该超分子细胞载体克服了现有技术需要通过细胞对药物载体的内吞作用才能实现细胞对药物载体的负载的缺陷,该超分子细胞载体不会引发细胞毒性,具有生物正交性。
在本发明应用较佳的实施方式中,上述大环主体分子为环糊精(CD)、葫芦脲(CB)、杯芳烃、柱芳烃或冠醚;优选的,大环主体分子为β-环糊精。
冠醚可以是双环冠醚、三环冠醚、多环冠醚和杂冠醚中的任意一种。
上述大环主体分子与许多客体分子有着较高的结合常数,有助于提升主客体复合物在体内的稳定性。
大环主体分子位于细胞膜的外表面层,大环分子链接的磷脂端与细胞膜磷脂双分子层融合从而嵌入细胞膜中。
在本发明应用较佳的实施方式中,上述第一细胞选自巨噬细胞、中性粒细胞、红细胞、干细胞、淋巴细胞、树突细胞、血小板和脂肪细胞中的任意一种。
不同的细胞类型具有不同的生理功能,如免疫细胞的炎症趋向性和干细胞的归巢作用等,这些细胞不同的生理功能也赋予了相应细胞较强的内在靶向驱动力,因此可根据疾病的病理特点选择合适类型的细胞作为靶向递送载体。本发明提供的超分子细胞载体可以根据需要选择相应的第一细胞。
巨噬细胞可以是M1型或M2型巨噬细胞。淋巴细胞可以是T细胞、B细胞和NK细胞中的至少一种。
脂肪细胞可以是白色脂肪细胞或褐色脂肪细胞。
在本发明应用较佳的实施方式中,上述大环主体分子与客体分子的摩尔比为1-10:1-10;优选为1:1;
优选的,客体分子为金刚烷或二茂铁。
大环主体分子与客体分子在上述摩尔比下可以简单快速的实现超分子细胞载体的制备。
客体分子需与主体分子相匹配,在其他实施方式中也可以根据需要进行置换。
在本发明应用较佳的实施方式中,上述纳米粒子为脂质体、胶束、纳米凝胶、无机纳米粒和纳米囊中的至少一种。
优选的,第二细胞为肝细胞、干细胞、淋巴细胞、树突细胞、血小板和脂肪细胞或红细胞。
在其他实施方式中,所有表面能够被“DSPE-PEG-客体分子”嵌膜的细胞都可作为第二细胞。
脂质体有利于超分子细胞载体跨膜运输,通过相似的极性实现靶向药物的递送。第二细胞为肝细胞或红细胞有利于提升超分子细胞载体对于重大疾病的靶向治疗能力。
在本发明应用较佳的实施方式中,上述嵌膜材料为PEG-DMPE、PEG-DPPE、PEG-DSPE或PEG-CHOL。
PEG-DMPE即PEG-二肉豆蔻酰基磷脂酰乙醇胺,PEG-DPPE即PEG-二棕榈酰基磷脂酰乙醇胺,PEG-DSPE即PEG-二硬脂酰基磷脂酰乙醇胺,PEG-CHOL即PEG-胆固醇。
在一种实施方式中,可以利用DSPE-PEG-ADA、胆固醇和卵磷脂为制备出表面富含ADA(金刚烷)的脂质体。
在另一种实施方式中,可以利用DSPE-PEG-ADA的嵌膜作用构建表面修饰客体分子金刚烷的肝细胞。
一种超分子细胞载体的制备方法,其包括:将偶联有嵌膜材料的大环主体分子与第一细胞共孵育得到第一部分,再将修饰有客体分子的纳米粒子或修饰有客体分子的第二细胞与第一部分混合孵育。
本发明提供的超分子细胞载体的制备方法,制备工艺简单、快速、条件温和、具有普适性,且该方法具有生物正交性。
在本发明应用较佳的实施方式中,上述制备方法还包括先将大环主体分子偶联嵌膜材料,再将偶联有嵌膜材料的大环主体分子嵌入第一细胞的细胞膜中;
优选的,大环主体分子与嵌膜材料中的PEG以共价键连接;优选的,偶联有嵌膜材料的大环主体分子与第一细胞共孵育的时间大于30分钟;偶联有嵌膜材料的大环主体分子的浓度为1μM-1mM。
在其他实施方式中,也可以直接购买偶联嵌膜材料的大环主体分子或修饰有客体分子的纳米粒子。
在本发明应用较佳的实施方式中,上述修饰有客体分子的纳米粒子或修饰有客体分子的第二细胞与第一部分混合孵育的时间≥10秒。
一种载药体系,载体体系包括超分子细胞载体和药物,药物装载在纳米粒子中或第二细胞中;优选的,纳米粒子为脂质体。
本发明提供的超分子细胞载体,可以用来递送脂质体或者细胞。药物可以装载在脂质体中,也就是作为纳米药物被细胞拉着靶向递送,其释放机制也主要涉及到脂质体本身的性质。载药脂质体脱离超分子细胞载体可以是如下几种方式:一是由于细胞膜的流动性所致脱落,二是载药脂质体被载体细胞或者靶组织细胞直接吞噬消化,造成胞内药物释放。
本发明提供的载药体系可以用于负载抗炎药物、抗生素、靶向癌症、神经系统疾病治疗剂。
抗炎药物可以是斛皮素,将抗炎药物斛皮素负载在脂质体中,与巨噬细胞超分子共轭后靶向递送到肺炎部位治疗急性肺炎。
载药体系可以用于负载阿霉素。
本发明具有以下有益效果:
本发明提供了一种超分子细胞载体、载药体系及其制备方法。基于超分子主客体作用构建的超分子细胞载体可实现基于细胞功能的靶向递送效果,具有高生物相容性、高生理屏障透过性和高靶向性。不需要对细胞表面进行共价键修饰,对修饰的细胞生理功能没有影响。本发明提供的超分子细胞载体的制备方法具有制备工艺简单、快速、条件温和和普适性的优势,该方法具有生物正交性。此外,还提供了一种载药体系,可以实现靶向药物治疗的载药。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,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相关的附图。
图1为本发明实施例1的超分子细胞与脂质体结合体的荧光成像图;
图2为本发明实施例1的超分子细胞与脂质体结合体的扫描电镜图;
图3为本发明实施例5的超分子细胞与细胞结合体的荧光成像图;
图4为本发明实施例5的超分子细胞与细胞结合体的扫描电镜图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。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。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本实施例提供了一种超分子细胞载体及其制备方法,本实施例中,第一细胞为巨噬细胞,DSPE-PEG-β-CD和DSPE-PEG-ADA均购自西安瑞禧生物科技有限公司,DMEM培养基购自赛默飞世尔科技(中国)有限公司,阿霉素购自上海阿拉丁生化科技股份有限公司。
将巨噬细胞在含有10μM的DSPE-PEG-β-CD的空白DMEM培养基中于37℃孵育2小时。
孵育后,洗去多余的DSPE-PEG-β-CD,再加入10μM的DSPE-PEG-ADA修饰的负载阿霉素的脂质体继续孵育2分钟。
洗去未结合的脂质体后,制得超分子细胞-脂质体结合体。
由于阿霉素有红色荧光,将制得的超分子细胞-脂质体结合体进行荧光成像和扫描电镜成像。超分子细胞与脂质体结合体的荧光成像图参照图1所示,超分子细胞与脂质体结合体的扫描电镜图参照图2所示。
实施例2
本实施例提供了一种超分子细胞载体及其制备方法,本实施例中,DSPE-PEG-β-CD和DMEM培养基与实施例1购买来源相同。本实施例中,第一细胞为巨噬细胞,将巨噬细胞在含有50μM的DSPE-PEG-β-CD的空白DMEM培养基中于37℃孵育1小时。
洗去多余的DSPE-PEG-β-CD后,加入50μM的DSPE-PEG-ADA修饰的脂质体继续孵育2分钟。最后洗去未结合的脂质体后,获得超分子细胞-脂质体结合体。
实施例3
本实施例提供了一种超分子细胞载体及其制备方法,DMPE-PEG-CB[7]和DMPE-PEG-ADA为实验室自制。本实施例中,将中性粒细胞在含有100μM的DMPE-PEG-CB[7](CB[7]为葫芦[7]脲)的空白DMEM培养基中于37℃孵育2小时。
然后洗去多余的DMPE-PEG-CB[7],加入100μM的DMPE-PEG-ADA修饰的脂质体继续孵育1分钟,洗去未结合的脂质体后。获得超分子细胞-脂质体结合体。
实施例4
本实施例提供了一种超分子细胞载体及其制备方法,DPPE-PEG-CB[7]和DPPE-PEG-ADA为实验室自制。本实施例中,将造血干细胞在含有40μM的DPPE-PEG-CB[7]的空白DMEM培养基中于37℃孵育1.5小时。
然后洗去多余的DPPE-PEG-CB[7],加入80μM的DPPE-PEG-ADA修饰的脂质体继续孵育5分钟,洗去未结合的脂质体后。获得超分子细胞-脂质体结合体。
实施例5
本实施例提供了一种超分子细胞载体及其制备方法,DiD和DiO均购自上海碧云天生物技术有限公司。本实施例中,将巨噬细胞在含有10μM的DSPE-PEG-β-CD的空白DMEM培养基中于37℃孵育2小时,在洗去多余的DSPE-PEG-β-CD后加入10μM的DSPE-PEG-ADA修饰的人体肝细胞继续孵育2分钟,洗去未结合的肝细胞后,制得超分子细胞-细胞结合体。
将获得的超分子细胞-细胞结合体进行荧光成像和扫描电镜成像。荧光成像图参照图3所示,扫描电镜成像图参照图4所示。其中巨噬细胞采用DiD(红色)染色,人体肝细胞采用DiO(绿色)染色。
实施例6
本实施例提供了一种超分子细胞载体及其制备方法,Fc购自上海阿拉丁生化科技股份有限公司,DSPE-PEG-Fc为实验室自制。本实施例中,将巨噬细胞在含有10μM的DSPE-PEG-β-CD的空白DMEM培养基中于37℃孵育2小时。孵育后洗去多余的DSPE-PEG-β-CD,并加入10μM的DSPE-PEG-Fc(Fc为二茂铁)修饰的人体肝细胞继续孵育2分钟,洗去未结合的细胞后,即获得超分子细胞-细胞结合体。
实施例7
本实施例提供了一种超分子细胞载体及其制备方法,DMPE-PEG-P5为实验室自制。本实施例中,将中心粒细胞在含有60μM的DMPE-PEG-P5(P5为柱[5]芳烃)的空白DMEM培养基中于37℃孵育2小时。孵育后,洗去多余的DMPE-PEG-P5,然后加入30μM的DSPE-PEG-Fc(Fc为二茂铁)修饰的红细胞继续孵育2分钟,洗去未结合的细胞后,获得细胞-细胞结合体。
实施例8
本实施例提供了一种超分子细胞载体及其制备方法,造血干细胞来源美国模式培养物集存库(ATCC),DPPE-PEG-β-CD和DPPE-PEG-ADA为实验室自制。本实施例中,将造血干细胞在含有100μM的DPPE-PEG-β-CD的空白DMEM培养基中于37℃孵育2小时,在洗去多余的DPPE-PEG-β-CD后加入150μM的DPPE-PEG-ADA修饰的红细胞继续孵育2分钟,洗去未结合的细胞后,获得细胞-细胞结合体。
对比例
将胚胎干细胞在含有10μM的DPPE-PEG-CB[7]的空白DMEM培养基中于37℃孵育5分钟,在洗去多余的DPPE-PEG-CB[7]后加入10μM DPPE-PEG-ADA修饰的载阿霉素脂质体继续孵育5分钟,洗去未结合的脂质体后,进行荧光成像,未发现胚胎细胞膜上有红色荧光。
综上所述,本发明实施例的超分子细胞载体是通过主客体作用相互连接形成的,是基于超分子主客体作用构建的新一代细胞制剂,可实现基于细胞功能的靶向递送效果;该超分子细胞载体的制备方法是将大环主体分子偶联嵌膜材料并嵌入第一细胞的细胞膜中;将客体分子修饰在纳米粒子表面或者第二细胞的细胞膜上;充分混合后即得超分子细胞载体。制备工艺简单、快速、条件温和、具有普适性和生物正交性,不需要对细胞表面进行共价键修饰,对修饰的细胞生理功能没有影响。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种超分子细胞载体,其特征在于,其包括通过主客体作用相互连接的第一部分和第二部分,所述第一部分为大环主体分子修饰的第一细胞,所述第二部分为客体分子修饰的纳米粒子或客体分子修饰的第二细胞,所述第一部分中的大环主体分子通过偶联嵌膜材料嵌入所述第一细胞的细胞膜中,所述大环主体分子与所述客体分子相对应。
2.根据权利要求1所述的超分子细胞载体,其特征在于,所述大环主体分子为环糊精、葫芦脲、杯芳烃、柱芳烃或冠醚;优选的,所述大环主体分子为β-环糊精。
3.根据权利要求2所述的超分子细胞载体,其特征在于,所述第一细胞选自巨噬细胞、中性粒细胞、红细胞、干细胞、淋巴细胞、树突细胞、血小板和脂肪细胞中的任意一种。
4.根据权利要求1所述的超分子细胞载体,其特征在于,所述大环主体分子与客体分子的摩尔比为1-10:1-10;优选为1:1;
优选的,所述客体分子为金刚烷或二茂铁。
5.根据权利要求1所述的超分子细胞载体,其特征在于,所述纳米粒子为脂质体、胶束、纳米凝胶、无机纳米粒和纳米囊中的至少一种;
优选的,所述第二细胞为肝细胞、干细胞、淋巴细胞、树突细胞、血小板和脂肪细胞或红细胞。
6.根据权利要求5所述的超分子细胞载体,其特征在于,所述嵌膜材料为PEG-DMPE、PEG-DPPE、PEG-DSPE或PEG-CHOL。
7.一种如权利要求1-6任一项所述的超分子细胞载体的制备方法,其特征在于,其包括:将偶联有嵌膜材料的大环主体分子与第一细胞共孵育得到第一部分,再将修饰有客体分子的纳米粒子或修饰有客体分子的第二细胞与第一部分混合。
8.根据权利要求7所述的超分子细胞载体的制备方法,其特征在于,所述制备方法还包括先将大环主体分子偶联嵌膜材料,再将偶联有嵌膜材料的大环主体分子嵌入第一细胞的细胞膜中;
优选的,所述大环主体分子与所述嵌膜材料中的PEG以共价键连接;优选的,偶联有嵌膜材料的大环主体分子与第一细胞共孵育的时间大于30分钟;所述偶联有嵌膜材料的大环主体分子的浓度为1μM-1mM。
9.根据权利要求7所述的超分子细胞载体的制备方法,其特征在于,修饰有客体分子的纳米粒子或修饰有客体分子的第二细胞与第一部分混合孵育的时间≥10秒。
10.一种载药体系,其特征在于,所述载药体系包括权利要求1-6任一项所述的超分子细胞载体和药物,所述药物装载在所述纳米粒子中或所述第二细胞中;优选的,所述纳米粒子为脂质体。
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