CN112089849B - 一种靶向脑胶质瘤的治疗基因递送载体和递送系统 - Google Patents
一种靶向脑胶质瘤的治疗基因递送载体和递送系统 Download PDFInfo
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Abstract
本发明提供了一种靶向脑胶质瘤的治疗基因递送载体和递送系统。该治疗基因递送载体包括:靶向分子,靶向分子包括脑主动靶向分子和细胞穿膜肽;载体骨架,载体骨架包括具有还原敏感性的双亲性共聚物、功能化磷脂和阳离子脂质且形成疏水性内核,功能化磷脂的一端嵌入载体骨架的亲水端、另一端与靶向分子连接,阳离子脂质的一端嵌入载体骨架的亲水端、另一端用于连接治疗基因;Fe3O4磁靶向,Fe3O4磁靶向包覆于载体骨架的疏水性内核中。基于具有还原敏感性的双亲性共聚物的还原敏感性有效释放运载的治疗基因;靶向分子能够实现跨血脑屏障和靶向胶质瘤细胞的双重作用,两种靶向分子组合Fe3O4磁靶向使得靶向效率又得到进一步提升。
Description
技术领域
本发明涉及脑胶质瘤的靶向治疗技术领域,具体而言,涉及一种靶向脑胶质瘤的治疗基因递送载体和递送系统。
背景技术
脑胶质瘤是最常见的中枢神经系统肿瘤,约占颅内肿瘤的46%。目前针对脑胶质瘤的治疗方式主要是手术切除,但由于其呈浸润型生长,使得肿瘤和正常脑组织边界不清,手术时难以完全切除,常常会导致复发。因此期望能够开发一种治疗方法,弥补手术难以完全清除病灶的缺点,高效治疗脑胶质瘤。现如今基因治疗被认为是一种有效的治疗手段,通过操作遗传物质来干预疾病的发生、发展和进程,包括替代或纠正人自身基因结构或功能上的错乱,杀灭病变的细胞或增强机体清除病变细胞的能力等,从而达到治病的目的。但是由于血脑屏障(Blood Brain Barrier,BBB)的存在,在防止有害物质进入脑,为脑组织提供相对稳定的内部环境,以维持其正常功能的同时也使治疗基因难以进入大脑,大大增加了治疗脑胶质瘤的难度。虽然各种病毒载体已被证明能够将基因高效传递到细胞中,但是由于其生物学特异性,例如免疫原性和毒性,导致其应用受到限制。相反,具有低免疫原性和相对安全性的非病毒载体引起了广泛的关注。因此,研究一种新的安全、有效的基因传递系统,能够跨过血脑屏障靶向脑胶质瘤是我们所期望的。
受体介导的转运蛋白在脑内皮细胞中高度表达,包括胰岛素受体、转铁蛋白受体蛋白1(TfR1)和低密度脂蛋白受体相关蛋白1(LRP1),这些高表达特异性配体为脑主动靶向提供了靶点。因此,可以借助这些特异性受体的配体达到脑主动靶向的目的。此外,通过在载体表面修饰细胞穿膜肽(CPP)也是提高穿透BBB和神经胶质瘤细胞效率的常用方法。CPP可以克服质膜的亲脂性障碍,穿透生物膜屏障,从而大大提高了将治疗基因传递到活细胞中的能力。
但是,上述技术的运用对于提高治疗基因的靶向穿透能力还有待提高。
发明内容
本发明的主要目的在于提供一种靶向脑胶质瘤的治疗基因递送载体和递送系统,以进一步提高基因传递系统的靶向胶质瘤能力。
为了实现上述目的,根据本发明的一个方面,提供了一种靶向脑胶质瘤的治疗基因递送载体,包括:靶向分子,靶向分子包括脑主动靶向分子和细胞穿膜肽;载体骨架,载体骨架包括具有还原敏感性的双亲性共聚物、功能化磷脂和阳离子脂质且形成疏水性内核,功能化磷脂的一端嵌入载体骨架的亲水端、另一端与靶向分子连接,阳离子脂质的一端嵌入载体骨架的亲水端、另一端用于连接治疗基因;Fe3O4磁靶向,Fe3O4磁靶向包覆于载体骨架的疏水性内核中。
进一步地,上述具有还原敏感性的双亲性共聚物、功能化磷脂和阳离子脂质摩尔比为(1~20):1:(1~20),优选为(3~7):1:(3~7),更优选为5:1:5。
进一步地,上述双亲性共聚物为PCL-ss-PEG-ss-PCL,优选双亲性共聚物的分子量为10000~75000,进一步优选为10000~20000,更优选双亲性共聚物为PCL3750-ss-PEG7500-ss-PCL3750。
进一步地,上述功能化磷脂为DSPE-PEG-COOH、DSPE-PEG-NHS、DSPE-PEG-Mal、DSPE-PEG-FITC、DSPE-PEG-NH2、DSPE-PEG-SH、DSPE-MPEG和DPPE-MPEG中的任意一种,优选为DSPE-PEG-Mal,优选分子量为2800~3000的DSPE-PEG-Mal,进一步优选DSPE-PEG-Mal为DSPE-PEG2000-Mal。
进一步地,上述阳离子脂质为DOTMA、DOTAP、DOSPA、DTAB、TTAB、CTAB、DDAB、DORI、DORIE、DORIE-HP、DORIE-HB、DORIE-HPc、DPRIE、DSRIE和DMRIE中的任意一种,优选为DOTAP。
进一步地,上述功能化磷脂、脑主动靶向分子、细胞穿膜肽和Fe3O4磁靶向的摩尔比为(1~4):(1~3):1:(10~30)。
根据本发明的另一方面,提供了一种靶向脑胶质瘤的治疗基因递送系统,包括载体和负载在载体上的治疗基因,该载体为上述任一种的递送载体。
进一步地,上述治疗基因为报告基因和/或脑胶质瘤治疗基因,优选为质粒DNA,siRNA或miRNA。
进一步地,上述递送载体与治疗基因摩尔比为(2.5~40):1,优选为(5~10):1,进一步优选为5:1。
进一步地,上述治疗基因递送系统的粒径为200~400nm。
应用本发明的技术方案,上述形成治疗基因递送载体的靶向分子、载体骨架和Fe3O4磁靶向共混后能够自组装形成内核包含Fe3O4的磷脂-聚合物纳米胶束;且基于具有还原敏感性的双亲性共聚物的还原敏感性,治疗基因递送载体能够在肿瘤细胞内谷胱甘肽(GSH)浓度下裂解,有效释放运载的治疗基因;使用脑主动靶向分子为第一靶向分子,细胞穿透肽为第二靶向分子,能够实现跨血脑屏障和靶向胶质瘤细胞的双重作用,经双分子修饰使递送系统的靶向效率相对于未修饰得到大大提升;在载体表面应用两种靶向分子的同时添加了Fe3O4磁靶向,上述三种靶向共同作用使得靶向效率由双分子修饰后的效率又得到进一步提升。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。在附图中:
图1为本申请一种实施例提供的靶向脑胶质瘤的治疗基因递送系统的结构示意图;
图2为Ang-TAT-Fe3O4-pEGFP-PCL-ss-PEG-ss-PCL LPNPs在不同N/P比例下的琼脂糖凝胶电泳结果;
图3为不同修饰的磷脂杂化聚合物纳米胶束在分散体系中的稳定性;
图4为不同浓度Ang-TAT-Fe3O4-pEGFP-PCL-ss-PEG-ss-PCL LPNPs下hCMEC/D3细胞(A)和C6细胞(B)的存活率;
图5A示出了实施例3中用Transwell和hCMEC/D3细胞建立体外BBB模型结构图;
图5B示出了实施例3的BBB模型的跨内皮电阻检测结果图;
图5C示出了实施例3的不同修饰的LPNPs穿透BBB后的跨内皮电阻值检测结果图;
图5D示出了实施例3中hCMEC/D3细胞培养时形成紧密连接构成离体BBB模型的CLSM图像;
图5E示出了实施例3中测定不同修饰的LPNPs组的BBB穿透率结果图,其中*表示与Ang-TAT-Fe3O4-pEGFP-PCL-ss-PEG-ss-PCL LPNPs组对比P<0.05;
图6A示出了实施例4中将hCMEC/D3细胞及C6细胞共培养构建的多靶标模型结构图,该模型放置在磁场上;
图6B示出了实施例4中将游离pEGFP-C1和具有不同修饰的LPNPs加入到构建完毕的多靶标模型中,待转染24小时后,在CLSM下检测转染C6细胞的荧光图像;
图6C示出了实施例4中将游离pEGFP-C1和具有不同修饰的LPNPs加入到构建完毕的多靶标模型中,待转染24小时后,通过流式细胞仪检测转染C6细胞的能力曲线图;
图6D示出了实施例4中游离pEGFP-C1和具有不同修饰的LPNPs通过BBB模型转染C6细胞的能力对比图,其中,**表示P<0.001,横线两端点所对应的组进行对比;
图7示出了本申请实施例所负载的pEGFP-C1质粒的结构示意图。
具体实施方式
需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
如背景技术所分析的,现有技术中脑主动靶向分子和细胞穿膜肽虽然可以在一定程度上提高治疗基因传递到活细胞中的能力,但是根据其治疗结果反馈,治疗基因的靶向穿透能力还有待提高。为了解决该问题,本申请提供了一种靶向脑胶质瘤的治疗基因递送载体和递送系统。
在本申请一种典型的实施方式中,提供了一种靶向脑胶质瘤的治疗基因递送载体,该治疗基因递送载体包括靶向分子、载体骨架和Fe3O4磁靶向,靶向分子包括脑主动靶向分子和细胞穿膜肽;载体骨架包括具有还原敏感性的双亲性共聚物、功能化磷脂和阳离子脂质且形成疏水性内核,功能化磷脂的一端嵌入载体骨架的亲水端、另一端与靶向分子连接,阳离子脂质的一端嵌入载体骨架的亲水端、另一端用于连接治疗基因;Fe3O4磁靶向包覆于载体骨架的疏水性内核中。
还原敏感性的双亲性共聚物的具有还原敏感性的连接基团,在细胞外环境下的优异稳定性和细胞内还原条件下的快速降解,因此可以在细胞外液中结合和保护核酸,在细胞内有效释放核酸。功能化磷脂能够通过其基团连接靶向分子和细胞穿膜肽,阳离子脂质可以凭借表面正电荷通过静电作用吸附携带负电荷的治疗基因。三种物质的结合能够组装成具有疏水性内核的载体骨架,借助于各自的性质,使得载体骨架具有包载及连接靶向分子、携带治疗基因、保护并实现细胞内释放基因的功能。同时将Fe3O4磁靶向包覆于载体骨架的疏水性内核中,不仅在磁场中可以发挥靶向作用,而且可以无创方式发挥出色的磁共振成像(MRI)性能。
上述形成治疗基因递送载体的靶向分子、载体骨架和Fe3O4磁靶向共混后能够自组装形成内核包含Fe3O4的磷脂-聚合物纳米胶束;且基于具有还原敏感性的双亲性共聚物的还原敏感性,治疗基因递送载体能够在肿瘤细胞内谷胱甘肽(GSH)浓度下裂解,有效释放运载的治疗基因;使用脑主动靶向分子为第一靶向分子,细胞穿透肽为第二靶向分子,能够实现跨血脑屏障和靶向胶质瘤细胞的双重作用,经双分子修饰使递送系统的靶向效率相对于未修饰得到大大提升;在载体表面应用两种靶向分子的同时添加了Fe3O4磁靶向,上述三种靶向共同作用使得靶向效率由双分子修饰后的效率又得到进一步提升。
如前所述,上述具有还原敏感性的双亲性共聚物、功能化磷脂和阳离子脂质各司其职且相辅相成,为了提高综合靶向作用和递送效率,优选上述具有还原敏感性的双亲性共聚物、功能化磷脂和阳离子脂质摩尔比为(1~20):1:(1~20),优选为(3~7):1:(3~7),更优选为5:1:5。
通常具有还原敏感性的基团包括二硫键等,为了使载体骨架的双亲性能和还原敏感性得到充分发挥,并控制治疗基因递送载体粒径,优选上述双亲性共聚物为PCL-ss-PEG-ss-PCL,优选双亲性共聚物的分子量为10000~75000,进一步优选为10000~20000,更优选双亲性共聚物为PCL3750-ss-PEG7500-ss-PCL3750。
用于本申请的功能化磷脂可以采用本领域中常用的功能化磷脂,包括但不限于DSPE-PEG-COOH、DSPE-PEG-NHS、DSPE-PEG-Mal、DSPE-PEG-FITC、DSPE-PEG-NH2、DSPE-PEG-SH、DSPE-MPEG、DPPE-MPEG。为了提高功能化磷脂与双亲性共聚物和靶向分子连接的稳定性,并控制治疗基因递送载体粒径,优选上述功能化磷脂为具有活性基团的DSPE-PEG-Mal,进一步优选分子量为2800~3000的DSPE-PEG-Mal,更优选DSPE-PEG-Mal为DSPE-PEG2000-Mal。
此外,用于本申请的阳离子脂质也可以从现有技术中常用的阳离子脂质中进行选择,比如氯化三甲基-2,3-二油烯氧基丙基铵(DOTMA)、溴化三甲基-2,3-二油酰氧基丙基铵(DOTAP)、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵(DOSPA)、溴化三甲基十二烷基铵(DTAB)、溴化三甲基十四烷基铵(TTAB)、溴化三甲基十六烷基铵(CTAB)、溴化二甲基双十八烷基铵(DDAB)、溴化二甲基-2-羟乙基-2,3-二油酰氧基丙基铵(DORI)、溴化二甲基-2-羟乙基-2,3-二油烯氧基丙基铵(DORIE)、溴化二甲基-3-羟丙基-2,3-二油烯氧基丙基铵(DORIE-HP)、溴化二甲基-4-羟丁基-2,3-二油烯氧基丙基铵(DORIE-HB)、溴化二甲基-5-羟戊基-2,3-二油烯氧基丙基铵(DORIE-HPc)、溴化二甲基-2-羟乙基-2,3-双十六烷氧基丙基铵(DPRIE)、溴化二甲基-2-羟乙基-2,3-双十八烷氧基丙基铵(DSRIE)、溴化二甲基-2-羟乙基-2,3-双十四烷氧基丙基铵(DMRIE)等,常用辅助脂质为辅助脂质主要有磷脂酰乙醇胺(PE)、磷脂酰胆碱(PC)、胆固醇(Chol)、二油酰基磷脂酰乙醇胺(DOPE)等。为提高和本申请所使用的治疗基因的连接稳定性,优选上述阳离子脂质为DOTAP。
在一种实施例中,为了进一步提高靶向能力,优选上述功能化磷脂、脑主动靶向分子、细胞穿膜肽和Fe3O4磁靶向的摩尔比为(1~4):(1~3):1:(10~30)。
此外,本申请对脑主动靶向分子和细胞穿膜肽没有特别要求,本领域中常用的脑主动靶向分子和细胞穿膜肽均可应用于本申请中,在此不再一一列举。Fe3O4磁靶向为本领域常规产品,通常为油酸修饰的Fe3O4磁性纳米粒等,具体也不一一列举。
在本申请另一种典型的实施方式中,提供了一种靶向脑胶质瘤的治疗基因递送系统,如图1所示,该治疗基因递送系统包括载体和负载在载体上的治疗基因,该载体为上述任一种的递送载体。
将治疗基因负载在上述递送载体上之后,基于具有还原敏感性的双亲性共聚物的还原敏感性,治疗基因递送载体能够在肿瘤细胞内谷胱甘肽(GSH)浓度下裂解,有效释放运载的治疗基因;使用脑主动靶向分子为第一靶向分子,细胞穿透肽为第二靶向分子,能够实现跨血脑屏障和靶向胶质瘤细胞的双重作用,经双分子修饰使递送系统的靶向效率相对于未修饰得到大大提升;在载体表面应用两种靶向分子的同时添加了Fe3O4磁靶向,上述三种靶向共同作用使得靶向效率由双分子修饰后的效率又得到进一步提升。
上述递送系统中的治疗基因可以为报告基因和/或脑胶质瘤治疗基因,优选为质粒DNA,siRNA或miRNA。
如前所述阳离子脂质和治疗基因以静电吸附的作用进行结合,为了提高治疗基因的吸附效率,优选上述递送载体与治疗基因摩尔比(即下文的N/P)为(2.5~40):1,优选为(5~10):1,进一步优选为5:1。其中递送载体的摩尔数按照本领域常规的计算方式进行计量,即以n=m/M,n为摩尔数,m为递送载体的质量,M为递送载体的分子量。
Fe3O4磁靶向的使用在一定程度上会提高治疗基因递送系统的粒径大小,本申请可以通过控制载体的分子量、用量来控制治疗基因递送系统的粒径,优选该粒径为200~400nm。
以下对本申请的治疗基因递送系统的制备方法进行举例说明,其中:
还原敏感性的双亲性共聚物为PCL-ss-PEG-ss-PCL,功能化磷脂为具有活性基团的DSPE-PEG-Mal,阳离子脂质为DOTAP,脑主动靶向分子为Ang,细胞穿膜肽为TAT。治疗基因可以自行提取,扩增或由商业公司提供。
制备方法包括:
将PCL-ss-PEG-ss-PCL、DSPE-PEG-Mal、DOTAP及Fe3O4以摩尔比为(1~20):1:(1~20):(10~30)的比例加入到茄形瓶中,加入5mL二氯甲烷,混合均匀,将茄形瓶安装到旋转蒸发仪上,瓶底与水浴装置接触,加热到37℃,以120rpm、580kPa旋转蒸发铺膜1h,待瓶内液体完全蒸干后将旋蒸仪调至真空,继续干燥1h,取下茄形瓶置于干燥器中干燥过夜。
过夜后,加入5mlL HEPES缓冲液,65℃下水化5h。
恢复室温后,冰浴超声混匀(每超声5min休息1-2min,5mm探头,Ampl30%)。
将超声后的载体使用PBS透析2h,期间更换3次透析液,形成Fe3O4-PCL-ss-PEG-ss-PCL LPNPs。
按照Ang与TAT摩尔比为(1~3):1的比例加入Ang-cys及TAT-cys后,加入2μL三乙胺,4℃震荡过夜,形成Ang-TAT-Fe3O4-PCL-ss-PEG-ss-PCL LPNPs,即递送载体。
静电吸附治疗基因:按照N/P=(2.5~40):1比例,分别加入合适量基因混匀,震荡混匀一定时间,室温下放置。最终形成Ang-TAT-Fe3O4-pDNA-PCL-ss-PEG-ss-PCL LPNPs,即递送系统。
以下将结合实施例和对比例,进一步说明本申请的有益效果。
实施例1
(1)将20mg PCL3750-ss-PEG7500-ss-PCL3750、0.8mg DSPE-PEG2000-Mal、1.02mgDOTAP及1.0mg油酸修饰的Fe3O4(摩尔比:5:1:5:15,PCL3750-ss-PEG7500-ss-PCL3750的分子量为15000,DSPE-PEG2000-Mal的分子量为2893,其中PCL3750-ss-PEG7500-ss-PCL3750按照常规方法合成;DSPE-PEG2000-Mal和DOTAP从纳米生物技术公司(中国北京)Nanocs BiologicalTechnology(Beijing,China)购买获得;油酸修饰的Fe3O4磁性纳米粒由西安瑞禧生物科技有限公司合成,所用为R-CY1010,粒径10nm)加入到5mL茄形瓶中,加入5mL二氯甲烷,混合均匀,将茄形瓶安装到旋转蒸发仪上,瓶底与水浴装置接触,加热到37℃,以120rpm、580kPa旋转蒸发铺膜1h,待瓶内液体完全蒸干后将旋蒸仪调至真空,继续干燥1h,取下茄形瓶置于干燥器中干燥过夜。
(2)过夜后,加入5mL HEPES缓冲液,65℃下水化5h。
(3)恢复室温后,冰浴超声10min(每超声5min休息1~2min,5mm探头,Ampl30%)。
(4)将超声后的载体使用PBS透析2h,期间更换3次透析液。收集制备好的载体Fe3O4-PCL-ss-PEG-ss-PCL LPNPs,待用。
(5)连接Ang及TAT。加入0.6mg Ang-cys、0.4mg TAT-cys(二者摩尔比1:1),加入2μL三乙胺,4℃震荡过夜,形成Ang-TAT-Fe3O4-PCL-ss-PEG-ss-PCL LPNPs。
(6)静电吸附pEGFP(该质粒结构见图7,为购自CLONTECH Laboratories.Inc.的Catalog#6084-1的pEGFP-C1质粒)。按照Ang-TAT-Fe3O4-PCL-ss-PEG-ss-PCL LPNPs和pEGFP的摩尔比(以N/P表示)为2.5/5/10/20/40比例,分别加入0.189mg、0.095mg、0.047mg、0.024mg、0.012mg pEGFP混匀,震荡10s混匀,室温下放置30min。形成5组磷脂杂化聚合物纳米胶束Ang-TAT-Fe3O4-pEGFP-PCL-ss-PEG-ss-PCL LPNPs(即递送系统),如图1所示。
(7)对(6)中具有不同N/P的5组磷脂杂化聚合物纳米胶束进行琼脂糖凝胶电泳,观察结果,确定合适的N/P比值。琼脂糖凝胶电泳过程如下:
①彻底清洗所有塑料实验用品。
②将凝胶成型模具放入成胶槽中,水平放置,将选好的梳子插入,梳子底部与模具之间留1mm空间。
③取0.25g琼脂糖于锥形瓶中,加入25mL去离子水(TAE buffer),加热(微波炉3min)至琼脂糖完全融化,取出待冷至70℃左右(手握可耐受)加入SYBR Green I 2.5μL(或者2.5μL SYBR Safe DNA Gel Stain),摇匀后缓缓倒入有机玻璃内槽,待胶凝固后(约20-30min),小心取出梳子和封端的透明胶,倒出电泳缓冲液。轻轻的将胶放在电泳槽内,加入电泳缓冲液(TAE buffer)使液面略高于胶面1mm。
④上述制备的具有不同N/P的5组磷脂杂化聚合物纳米胶束10μL(可稀释,如2μL样品稀释至20μL)加入1/5体积的上样缓冲液2μL(6×loading dye),短暂离心混合后上样10μL复合物(含DNA约300ng)。
⑤开始电泳,电压80V,电流100mA,时间25min左右(或50min左右待条带跑至距胶板边缘1-2cm左右)时于紫外下观察。
测定结果如图2所示,当N/P>5时,捕获的pEGFP会残留在孔中。此现象表明,当N/P>5时,带正电的DOTAP足以中和带负电的pEGFP,考虑到提高包封效率,我们认为当N/P=5时,为该聚合物纳米胶束运载治疗基因的最适N/P比值。
实施例2
比较不同修饰的磷脂杂化聚合物纳米胶束粒径、电位,稳定性和细胞毒性
将20mg PCL-ss-PEG-ss-PCL、0.8mg DSPE-PEG-Mal、1.02mg DOTAP加入到5mL茄形瓶中,加入5mL二氯甲烷,混合均匀,将茄形瓶安装到旋转蒸发仪上,瓶底与水浴装置接触,加热到37℃,以120rpm、580kPa旋转蒸发铺膜1h,待瓶内液体完全蒸干后将旋蒸仪调至真空,继续干燥1h,取下茄形瓶置于干燥器中干燥过夜。过夜后,加入5mL HEPES缓冲液,65℃下水化5h。恢复室温后,冰浴超声10min(每超声5min休息1-2min,5mm探头,Ampl30%)。将超声后的载体使用PBS透析2h,期间更换3次透析液。形成PCL-ss-PEG-ss-PCL LPNPs,待用。
对上述PCL-ss-PEG-ss-PCL LPNPs进行Ang修饰,加入0.6mg Ang-cys、加入2μL三乙胺,4℃震荡过夜,生成Ang-PCL-ss-PEG-ss-PCL LPNPs。
对上述PCL-ss-PEG-ss-PCL LPNPs进行Ang及TAT修饰,加入0.6mg Ang-cys、0.4mgTAT-cys、加入2μL三乙胺,4℃震荡过夜,生成Ang-TAT-PCL-ss-PEG-ss-PCL LPNPs。
按照N/P=5,对PCL-ss-PEG-ss-PCL LPNPs、Ang-PCL-ss-PEG-ss-PCL LPNPs、Ang-TAT-PCL-ss-PEG-ss-PCL LPNPs、以及实施例1的Ang-TAT-Fe3O4-PCL-ss-PEG-ss-PCL LPNPs进行静电吸附质粒pEGFP,加入0.095mg pEGFP混匀,震荡10s混匀,室温下放置30min。形成pEGFP-PCL-ss-PEG-ss-PCL LPNPs、Ang-pEGFP-PCL-ss-PEG-ss-PCL LPNPs、Ang-TAT-pEGFP-PCL-ss-PEG-ss-PCL LPNPs、Ang-TAT-Fe3O4-pEGFP-PCL-ss-PEG-ss-PCL LPNPs。
将载体骨架PCL-ss-PEG-ss-PCL LPNPs及四种不同修饰的磷脂杂化聚合物纳米胶束分散在PBS缓冲液中,得到五种均匀稳定的分散体系,并使用粒度仪测量不同载体的粒径和电位,结果如表1所示,Fe3O4的添加增加了LPNPs的粒径,但仍在安全范围内。此外,在全身生理pH值下,大脑内皮细胞的腔表面呈现整体负电荷,实验结果中Zeta电位为正性,能够促进转运的过程。
表1
磷脂杂化聚合物纳米胶束 | 粒径(nm) | Zeta电位(mV) |
PCL-ss-PEG-ss-PCL LPNPs | 84.55±0.27 | ﹣11.73±0.37 |
pEGFP-PCL-ss-PEG-ss-PCL LPNPs | 181.00±3.74 | 6.00±0.10 |
Ang-pEGFP-PCL-ss-PEG-ss-PCL LPNPs | 193.00±0.82 | 8.36±0.22 |
Ang-TAT-pEGFP-PCL-ss-PEG-ss-PCL LPNPs | 199.33±1.70 | 9.07±0.24 |
Ang-TAT-Fe<sub>3</sub>O<sub>4</sub>-pEGFP-PCL-ss-PEG-ss-PCL LPNPs | 302.33±3.68 | 4.66±0.15 |
将表1中后四种磷脂杂化聚合物纳米胶束分散在4℃的PBS磷酸缓冲盐溶液中,每天在同一时间检测其粒径变化,连续检测一周,结果如图3A所示,在一周内这些磷脂杂化聚合物纳米胶束的粒径没有明显变化,提示稳定性良好。
此外,通过以下方式进一步测定了不同修饰的LPNPs的工作稳定性:内皮细胞培养基(ECM,Gibco,Carlsbad,CA,USA)中添加10%胎牛血清(FBS,Gibco,Carlsbad,CA,USA)在37℃下观察粒径变化,测定不同修饰的LPNPs的工作稳定性,结果见图3B。
为明确载体中各组分对细胞是否有杀伤作用,将Ang-TAT-Fe3O4-pEGFP-PCL-ss-PEG-ss-PCL LPNPs以不同浓度(50μg/mL、100μg/mL、200μg/mL、300μg/mL、400μg/mL)分别加入到传代后生长良好的hCMEC/D3细胞和C6细胞中,在适宜细胞生长的条件下孵育24h,观察细胞的死亡情况。结果如图4所示,添加不同浓度的Ang-TAT-Fe3O4-pEGFP-PCL-ss-PEG-ss-PCL LPNPs的两种细胞在2h和24h的存活率均在95%以上。另外,添加不同浓度的Ang-TAT-Fe3O4-pEGFP-PCL-ss-PEG-ss-PCL LPNPs的两种细胞的2h存活率和24h存活率之间无显着差异(P>0.05)。这表明该基因传递系统对癌细胞系(C6)和人脑微血管内皮细胞系(hCMEC/D3)的细胞毒性很低。
实施例3
比较不同修饰的磷脂杂化聚合物纳米胶束的跨越BBB的能力
用Transwell和hCMEC/D3细胞建立体外BBB模型(图5A)。在Transwell 3460(Corning,USA)平板的上腔室中以4×105/孔的密度种植以第二代或第三代传代的hCMEC/D3细胞,Transwell平板的下腔室添加每天更换的ECM培养基,并放入板在37℃培养箱中。
BBB模型的跨内皮电阻(TEER)检测。如图5B所示,TEER值在第四天趋于稳定并超过130Ω·cm2。在不同的修饰的LPNPs穿透BBB之后,同样进行了BBB模型的TEER值检测,结果表明TEER无明显变化(图5C),意味着制备的LPNPs对BBB模型的完整性破坏很小。
观测hCMEC/D3细胞的紧密连接。当TEER值达到100Ω·cm2以上并稳定时,使用微丝绿色荧光探针和DAPI孔中染色后,观察hCMEC/D3细胞的CLSM图像。图5D显示hCMEC/D3细胞之间已经形成紧密连接。
测定不同修饰的LPNPs组的BBB穿透率。使用Cy5标记pEGFP,在2h,6h,12h和24h用荧光分光光度计半定量分析不同修饰的LPNPs组的BBB穿透率。如图5E所示,与其他LPNPs相比,Ang-TAT-Fe3O4-pEGFP-PCL-ss-PEG-ss-PCL LPNPs对BBB的渗透效果更好(P<0.05),并且随着培养时间的延长其透射率也增加。
实施例4
比较不同修饰的磷脂杂化聚合物纳米胶束跨越BBB后的转染能力
使用共聚焦显微镜和流式细胞分析仪定性和定量分析实施例2中所形成的四种不同修饰的磷脂杂化聚合物纳米胶束(以下称为纳米粒)及游离的质粒pEGFP跨越BBB后对C6细胞的转染能力。
(1)流式24孔板准备:
①用0.25%胰蛋白酶消化C6细胞(培养至第二、三代),再用10%胎牛血清的DMEM培养液配成单个细胞悬液,以(2~4)×105浓度1mL接种至24孔板中,使用含双抗培养基培养一天。
②过夜后,将培养皿中的培养基吸弃,每个培养皿加入2mL用培养基稀释的纳米粒(每个24孔板含DNA 0.05μg左右),培育4h,倒掉样品,于含血清、双抗的培养基中培育24h。
③内容设置:
浓度:基因每孔0.05μg、0.1μg、0.2μg,每孔四个平行,一个空白对照
④样品处理
a.用常温PBS洗涤2次后,用不含EDTA的胰酶消化收集后,于室温1000rpm离心5分钟,收集细胞;
b.将细胞轻柔重悬于500μL PBS中,过筛上机流式检测。若当天不能检测,可以用4%多聚甲醛500μL重悬,4℃保存。
c.过筛后1h内进行检测。
结果如图6A至图6D所示,游离pEGFP质粒几乎对C6细胞没有转染能力,单纯使用载体携带pEGFP质粒对C6细胞的转染能力很低,共聚焦显微镜下几乎肉眼不可见绿色荧光,流式细胞分析结果仅为1.51%,使用Ang修饰载体后转染能力提高,荧光在共聚焦显微镜下肉眼可见,转染率为5.20%,使用Ang和TAT共同修饰的载体荧光强度再次增强,转染率提高到8.23%,当再加上Fe3O4进行磁性靶向,转染率则能提高到14.6%。综上所述,表明两种靶向基团与Fe3O4磁靶向可以进行协同作用,共同帮助载体对细胞进行转染。
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)
1.一种靶向脑胶质瘤的治疗基因递送载体,其特征在于,包括:
靶向分子,所述靶向分子包括脑主动靶向分子和细胞穿膜肽;
载体骨架,所述载体骨架包括具有还原敏感性的双亲性共聚物、功能化磷脂和阳离子脂质且形成疏水性内核,所述功能化磷脂的一端嵌入所述载体骨架的亲水端、另一端与所述靶向分子连接,所述阳离子脂质的一端嵌入所述载体骨架的亲水端、另一端用于连接治疗基因,所述阳离子脂质为DOTAP;
Fe3O4磁靶向,所述Fe3O4磁靶向包覆于所述载体骨架的疏水性内核中,
所述具有还原敏感性的双亲性共聚物、所述功能化磷脂和所述阳离子脂质摩尔比为(1~20):1:(1~20),所述双亲性共聚物为PCL3750-ss-PEG7500-ss-PCL3750,所述功能化磷脂为DSPE-PEG2000-Mal,所述功能化磷脂、所述脑主动靶向分子、所述细胞穿膜肽和所述Fe3O4磁靶向的摩尔比为(1~4):(1~3):1:(10~30)。
2.根据权利要求1所述的治疗基因递送载体,其特征在于,所述具有还原敏感性的双亲性共聚物、所述功能化磷脂和所述阳离子脂质摩尔比为(3~7):1:(3~7)。
3.根据权利要求2所述的治疗基因递送载体,其特征在于,所述具有还原敏感性的双亲性共聚物、所述功能化磷脂和所述阳离子脂质摩尔比为5:1:5。
4.一种靶向脑胶质瘤的治疗基因递送系统,包括载体和负载在所述载体上的治疗基因,其特征在于,所述载体为权利要求1至3中任一项所述的递送载体。
5.根据权利要求4所述的治疗基因递送系统,其特征在于,所述治疗基因为报告基因和/或脑胶质瘤治疗基因。
6.根据权利要求5所述的治疗基因递送系统,其特征在于,所述治疗基因为质粒DNA,siRNA或miRNA。
7.根据权利要求4所述的治疗基因递送系统,其特征在于,所述递送载体与所述治疗基因摩尔比为(2.5~40):1。
8.根据权利要求4所述的治疗基因递送系统,其特征在于,所述递送载体与所述治疗基因摩尔比为(5~10):1。
9.根据权利要求4所述的治疗基因递送系统,其特征在于,所述递送载体与所述治疗基因摩尔比为5:1。
10.根据权利要求4所述的治疗基因递送系统,其特征在于,所述治疗基因递送系统的粒径为200~400nm。
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