CN117503949A - 脑靶向肽修饰的“杂合脂质体”纳米粒 - Google Patents
脑靶向肽修饰的“杂合脂质体”纳米粒 Download PDFInfo
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- CN117503949A CN117503949A CN202210896950.1A CN202210896950A CN117503949A CN 117503949 A CN117503949 A CN 117503949A CN 202210896950 A CN202210896950 A CN 202210896950A CN 117503949 A CN117503949 A CN 117503949A
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- liposome
- drug
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- exosome
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Abstract
本发明公开了一种脑靶向肽修饰的“杂合脂质体”纳米粒的制备方法和应用,所述纳米粒主要是由功能化脂质体和外泌体融合而成,所述功能化脂质体主要由脑靶向肽、磷脂和脂溶性活性药物构成。“杂合脂质体”纳米粒的制备方法是薄膜分散法。本发明制备工艺简单、条件温和、成本低廉,制得的纳米粒具有载药量高,载药种类多样、高BBB穿透性、生物靶向调控、生物安全性等多种优点。该纳米药物递送平台能够实现药物递送、联合给药以及协同精准治疗,能够实现对于复杂的进行性疾病如肿瘤、神经退行性疾病等的协同靶向治疗,具有良好的应用前景。
Description
技术领域
本发明涉及一种穿透血脑屏障(Blood brain barrier,BBB)的杂合脂质体纳米粒,具体涉及靶向肽、脂质体和外泌体融合的纳米粒,属于生物医药技术领域。
背景技术
脂质体是人工合成纳米材料,具有良好的生物降解性和相容性,大小可调控性好、药物密封性能优良、易于修饰,被广泛应用于纳米药物递送系统的设计。脂质体由脂质双分子层组成的,分子层间为水相的闭合囊泡,能够包载亲水或疏水性药物,提高药物的稳定性;脂质体具有高度亲脂性,可通过被动转运、内吞或与脑血管内皮细胞膜融合等途径转运至脑实质,用于药物的脑内递送。此外,通过在脂质体的脂质双分子层上修饰抗体、糖残基和受体配体等,可以赋予其特异主动靶向性,将药物输送到特定的组织器官、细胞或亚细胞器。经配体表面修饰的脂质体、纳米粒、胶束等纳米递药系统能够协助药物通过BBB,提高脑内的药物浓度,已成为目前脑部药物递送的重要手段之一。近年来,多肽在制药领域的应用受到研究届的关注。它们不仅具有生物活性,还擅长将货物运输到靶区,其在靶向治疗中的应用具有巨大的前景。RGD肽(arginine-glycine-aspartic acid)可以靶向整合素αvβ3,介导细胞粘附;血管内皮素2(angiopep-2)是一种19聚肽,其与大脑内皮细胞上低密度脂蛋白受体相关蛋白1结合后能够穿过BBB;Cereport(RMP7),是一种缓激肽类似物,可以特异性地与脑微血管内皮细胞上的B2缓激肽受体结合并有效地通过BBB。
近年来,仿生材料因具有自发的生命系统运行模式和生物材料结构规律而引起了人们的广泛关注。其中,外泌体是一种内源性、直径在40-150nm的胞外囊泡,各种类型的细胞均可分泌外泌体,其普遍存在于体液中,包括血液、尿液、唾液、脑脊液等。外泌体含有与细胞来源相关的蛋白质和RNA,参与细胞间通讯,可用于疾病的诊断、监测以及药物疗效的筛选。此外,外泌体向病变细胞递送活性物质方面的特性推动其作为治疗载体的研究。外泌体膜表面具有多种特性跨膜蛋白,如四跨膜蛋白超家族蛋白CD9,HSP70等。基于外泌体特征蛋白设计的递送系统能够靶向组织病区并发挥疾病治疗作用。外泌体具有良好的生物相容性,能够逃脱机体吞噬,且可以透过BBB。外泌体可特异性归巢其来源细胞并进行膜融合,而BBB表面广泛分布脑微血管内皮细胞,故此,以脑血管内皮细胞来源的外泌体与脂质体共建的脑靶向肽修饰的“杂合脂质体”纳米粒,将具有特异性归巢至BBB的能力,提高药物的入脑量。
神经退行性疾病是最具破坏性但却治疗不善的疾病。随着老年人口的增加,在未来20年,治疗脑部疾病的全球药物开发必须迅速增长。然而,由于缺乏有效的技术通过血脑屏障输送药物阻碍了神经退行性疾病药物的开发,亟需寻找高效的递药系统解决疾病难和疗效差的问题。
发明内容
针对现有技术的缺陷,以脑血管内皮细胞来源的外泌体与脂质体共建的外泌体-脂质体纳米靶向递药系统,将具有特异性归巢至BBB的能力,提高药物的入脑量。将脂质体与外泌体融合,开发外泌体-脂质体纳米靶向递药系统,协助药物跨越BBB,增加药物入脑量,可有效提高递药系统的稳定性,延长其体内血液循环半衰期,增加其在脑部的蓄积,从而提高神经退行性疾病治疗效果。
本发明的目的是拟利用脑毛细血管内皮细胞来源的外泌体特异性归BBB的作用,构建外泌体-脂质体纳米靶向递药系统,协助药物跨越BBB,增加药物入脑量;将具有开放BBB作用的缓激肽修饰该递药系统,增加药物入脑量;递药系统中包载血管扩张剂肼苯哒嗪,可降低胶质瘤血管的高压状态,增加化疗药物的灌注,通过以下技术方案实现:
一种脑靶向肽修饰的“杂合脂质体”纳米粒,其特征在于,所述“杂合脂质体”纳米粒主要是由功能化脂质体和外泌体融合而成,功能化脂质体主要由靶向肽、磷脂和脂溶性活性药物构成,是通过以下方法制备而成,具体包括如下步骤:
1)制备含有磷脂、Chol(胆固醇)、DSPE-PEG、DSPE-PEG-NHS、脂溶性活性药物的有机溶液A:
当磷脂质量为46.25mg时,有机溶剂质量以5mL计,固体质量比为磷脂:Chol:DSPE-PEG:DSPE-PEG-NHS:脂溶性活性药物=23.125:1.5:6.25:2:1.5;
2)旋转蒸发有机溶液A除去有机溶剂,PBS水化,超声破碎,过膜,得载药脂质体;
3)载药脂质体与靶向肽溶液孵育,得载药功能化脂质体:
其中,靶向肽溶液是将1mg靶向肽溶于1mL超纯水中得到,浓度为1mg/mL,靶向肽溶液与载药脂质体4℃孵育12h,所述靶向肽与DSPE-PEG-NHS的质量比=1:16;
4)提取分离获得外泌体:采用密度梯度离心法提取分离得到外泌体,转速为300g—100000g;
5)取步骤(3)所得载药功能化脂质体和步骤(4)所得外泌体挤出:其中,载药功能化脂质体以磷脂总量计,外泌体以蛋白含量计,所述载药功能化脂质体和载药外泌体质量比为5:1,使用挤出器将脂质体与外泌体挤出10-20次,收集,即得靶向肽修饰的“杂合脂质体”纳米粒。
所述步骤1)中的有机溶剂为甲醇,所述磷脂选自DOPC、DOPS、DOPE、DMPC、DMPE中的一种或几种,所述脂溶性活性药物为非瑟酮,吡格列酮,尼达尼布中的一种或几种。
所述步骤2)中的旋转蒸发时间为30min,水化时间为30min,超声破碎时间为5~10min。
所述步骤3)中的靶向肽为杭州佳肽生物公司合成,氨基酸序列为H-Arg-Pro-Hyp-Gly-Thi-SerPro-4-Me-Tyr(psi CH2NH)-Arg-OH。
本发明的优点:
(1)高度内源性:利用天然来源的外泌体与功能化脂质体融合,所得“杂合脂质体”纳米粒保留了外泌体的内源生理特性,具有高度内源性;
(2)高穿透性:纳米级别粒径,具有被动靶向性,易从血管内扩散至血管外;内源性外泌体及其具有的特异性蛋白受体使得其能够穿透生理屏障;靶向肽的修饰进一步增强其细胞以及组织的穿透能力;
(3)生物靶向调控:“杂合脂质体”纳米粒保留了外泌体具有的特异性蛋白受体,能够与病变细胞表面受体结合;通过靶向肽的修饰靶向病变部位细胞及其他高表达受体,具有生物靶向调控能力。同时,外泌体与功能化脂质体融合后,两者协同作用,显著提高靶向性;
(4)高生物安全性:“杂合脂质体”融合纳米粒具有良好的生物相容性,可生物降解,毒副作用低;
(5)治疗机制互补:外泌体和脂质体膜融合的方式构建的共载多组分融合纳米粒,能够实现联合给药及治疗机制互补;
(6)治疗手段多元化:通过修饰不同的靶向肽、装载不同的药物活性成分或其他诊断监测类成分,实现“杂合脂质体”纳米粒治疗疾病或诊断疾病的能力。
有益效果:与现有技术相比,本发明具有以下优势:
(1)本发明采用挤出方法进行“杂合脂质体”纳米粒的制备,方法简单、成本低廉;
(2)本发明提供的靶向肽修饰的“杂合脂质体”纳米粒可穿透生理屏障,与脑相关细胞及其他高表达受体特异性结合,具有主动靶向性;且纳米粒保留了内源性外泌体的生理特性,具有高度内源性和生物安全性;
(3)本发明提供的靶向肽修饰的“杂合脂质体”纳米粒能够通过装载不同治疗机制的活性药物达到联合给药、协同精准治疗的目的,为多药递送、多途径治疗疾病提供一种新的设计思路。
本发明提供的靶向肽修饰的“杂合脂质体”纳米粒靶向脑部并穿透血脑屏障(BBB),具有高度内源性、高穿透性、载药种类多样、生物靶向调控、高生物安全性、治疗机制互补、治疗手段多元化等特性,成功构建了仿生型“杂合脂质体”纳米递送系统,为神经退行性疾病的高效靶向治疗提供了范本,具有广阔的应用前景以及临床转化潜力。
附图说明:
图1外泌体表面特征蛋白的表达;
图2A为靶向肽修饰的“杂合脂质体”纳米粒的透射电镜图像,图2B为纳米粒的大小;
图3为靶向肽修饰的“杂合脂质体”纳米粒膜融合考察;
图4A为靶向肽修饰的“杂合脂质体”纳米粒的细胞摄取荧光图像,图4B为荧光定量;
图5为靶向肽修饰的“杂合脂质体”纳米粒跨BBB能力的考察。
具体实施方式
通过下列实施实例进一步说明本发明的技术方案,但本发明的保护范围,不局限于此。
实施例1:包载非瑟酮(FIS)的功能化脂质体(RMP7-L-FIS)的制备工艺
取磷脂46.25mg、Chol 3.0mg、DSPE-PEG 12.5mg、DSPE-PEG-NHS 4.0mg、FIS粉末3.0mg,溶于5mL甲醇中,超声5min,50℃旋转蒸发30min,挥干有机溶剂,成膜。加5mL PBS水化30min。水浴超声30min,探头超声5min(冰浴),过0.22μm滤膜,即得载药脂质体(L-FIS)。
将0.25mL RMP7溶液添加到上述载药脂质体溶液(L-FIS)中,同时轻轻搅拌(150rpm/min),使反应在4℃进行12h,即得载药功能化脂质体(RMP7-L-FIS)。
实施例2:提取分离获得细胞外泌体
无外泌体培液提取:无添加细胞培液加10%FBS;超速离心机,4℃120000g离心8h;收集上层培液,即为去除外泌体的培液;超净台,过0.22μm滤膜,除菌,加1%双抗。
外泌体提取:无外泌体培液培养细胞,收集细胞培液,在4℃下300g,10min,2000g,20min除去漂浮细胞及细胞碎片,收集上清。上清液在4℃下10000g,30min除去微囊泡等,收集上清液。过0.22μm滤膜。上清液在4℃下100000g,60min,获得沉淀即为外泌体。将收集到沉淀用PBS洗涤。再次在4℃下100000g,60min离心,回收沉淀。最终提取的外泌体重悬于50-200uL的灭菌PBS中,保存在-80℃,用BCA测定外泌体浓度。
实施例3:靶向肽修饰的“杂合脂质体”纳米粒的制备工艺
采用膜挤压法制备“杂合脂质体”纳米粒,使载药功能化脂质体(RMP7-L-FIS)和外泌体(E)发生膜融合(RMP7-EL-FIS),具体操作为取以蛋白含量计100μg的外泌体(实施例2)和以磷脂总量计500μg的载药功能化脂质体(RMP7-L-FIS)(实施例1),然后分别通过400和200nm聚碳酸酯膜挤出10次,即得“杂合脂质体”纳米粒(RMP7-EL-FIS)。
实施例4:靶向肽修饰的“杂合脂质体”纳米粒性质考察
1.1外泌体表面特征蛋白的表达
BCA法测定外泌体的蛋白质浓度:取外泌体溶液与RIPA裂解液等体积混匀,冰上反应15min,取反应液10μL,加10μL PBS溶液混匀,加入200μL BCA工作液,37℃恒温震荡30min,于562nm波长处测定吸光度值,根据标准曲线计算样品中蛋白浓度,保存在-20℃。
免疫印迹法检测外泌体标志蛋白(CD9、HSP70)和阴性标记(钙粘连蛋白/calnexin)。
结果如图1所示,CD9和HSP70均表达阳性,内质网标记物钙粘连蛋白缺失,证实外泌体表面特征蛋白的表达。
1.2靶向肽修饰的“杂合脂质体”纳米粒的形态
采用负染法置于透射电镜下观察靶向肽修饰的“杂合脂质体”纳米粒(RMP7-EL-FIS)的表面形态,马尔文粒径仪检测纳米粒的粒径,电位和PDI(多分散性指数)。
结果如图2A和图2B所示,透射电子显微镜分析显示纳米粒呈圆形,有明显的脂层,粒径~100nm,ζ电位是-3.8mV和PDI为0.22。
1.3靶向肽修饰的“杂合脂质体”纳米粒膜融合考察
1.3.1FRET荧光光谱扫描验证融合发生
称取磷脂23.125mg、DSPE-PEG 6.25mg、Chol 1.5mg、5uL NBD-PE、5uL RhB-PE和2.5mL甲醇,5min超声,50℃旋转蒸发30min,除去有机溶剂,成膜。加5mL PBS水化,50℃30min。水浴超声30min,探头超声5min(冰浴),过0.22μm滤膜,即得纳米粒NBD/RhB-L。
采用膜挤压法制备NBD/RhB-EL
利用荧光分光光度计设置NBD荧光分子的激发波长470nm,扫描450-700nm波长范围内纳米粒NBD/RhB-L的荧光曲线。结果如图3中NBD/RhB-L的荧光光谱曲线所示,当设置供体分子NBD的激发波长470nm去激发NBD分子,其产生530nm处的荧光能够激发RhB产生580nm的发射波长,存在FRET现象。当外泌体和NBD/RhB-L发生膜融合后(NBD/RhB-EL),由于NBD/RhB-L的磷脂双分子层遭到外泌体插入而稀释,磷脂双分子层中供体荧光分子NBD和受体荧光分子RhB的空间距离增加,FRET效率减弱,因此外泌体和NBD/RhB-L融合后纳米粒(EL)在530nm处的峰强度增加,在580nm处的峰强度降低,说明外泌体和NBD/RhB-L膜融合的发生。
1.4靶向肽修饰的“杂合脂质体”纳米粒的细胞摄取
取磷脂46.25mg、Chol 3mg、DSPE-PEG 12.5mg、DSPE-PEG-NHS 4.0mg,香豆素6(C6)4mg,Chol 3.0mg,溶于5mL甲醇中,超声5min,50℃旋转蒸发30min,除去有机溶剂,成膜。加5mL PBS水化30min。水浴超声30min,探头超声5min(冰浴),过0.22μm滤膜,即得荧光脂质体(C6-L)。将C6-L与0.25mg RMP7溶液中共孵育,4℃进行12h,即得荧光功能化脂质体(C6-RMP7-L)。参考实施例3采用膜挤压法制备荧光“杂合脂质体”纳米粒(C6-RMP7-EL)。
取对数生长的Bend.3小鼠脑微血管内皮细胞,1×105个细胞密度接种于24孔板中,37℃,5%CO2培养至细胞贴壁。细胞贴壁后,分别加入含有C6-L,C6-RMP7-L和C6-RMP7-EL的单培溶液培养0.5或4h。取出孔板,吸去培养液,PBS清洗后,加4%多聚甲醛溶液固定细胞20min,固定完毕,吸去多聚甲醛,PBS清洗两次,滴加含有DAPI的抗荧光猝灭封片剂进行封片后,荧光显微镜下观察细胞摄取纳米粒。
结果如图4A和图4B所示,“杂合脂质体”纳米粒(RMP7-EL)的绿色C6荧光信号在Bend.3细胞胞质内有高度显示,说明Bend.3细胞能够高效大量摄取靶向肽修饰的“杂合脂质体”纳米粒(RMP7-EL)。
1.5靶向肽修饰的“杂合脂质体”纳米粒的细胞毒性
1.5.1RMP7-EL-FIS-PIO的制备
取磷脂46.25mg、Chol 3.0mg、DSPE-PEG 12.5mg,DSPE-PEG-NHS 4.0mg,FIS粉末3.0mg和PIO粉末3.0mg溶于5mL甲醇中,超声5min,50℃旋转蒸发30min,除去有机溶剂,成膜。加5mL PBS水化30min。水浴超声30min,探头超声5min(冰浴),过0.22μm滤膜,即得双载药脂质体(L-FIS-PIO)。将0.25mL RMP7溶液添加到双载药脂质体溶液中,同时轻轻搅拌(150rpm/min),使反应在4℃进行12h,即得功能化双载药脂质体(RMP7-L-FIS-PIO),与外泌体挤出得RMP7-EL-FIS-PIO。
1.5.2纳米粒子的细胞毒性
取实施例三,四下所得RMP7-EL-FIS和RMP7-EL-FIS-PIO,HPLC测定其中FIS和PIO的含量,取FIS粉末1mg,加甲醇配制成浓度为1mg/mL的FIS溶液,取PIO粉末1mg,加甲醇配制成1mg/mL的PIO溶液。设置空白对照组、游离药物FIS、游离FIS+PIO、RMP7-EL-FIS、RMP7-EL-FI-PIO组。
取对数生长的SH-SY5Y神经母细胞瘤细胞,1×104个细胞密度接种于96孔板中,37℃,5%CO2培养至细胞贴壁。待细胞生长到80%左右时,弃上清液,加入用培液配制的一系列梯度浓度的PBS、游离药物FIS、游离FIS+PIO、RMP7-EL-FIS和RMP7-EL-FI-PIO,每孔100uL,每个浓度平行设置3个复孔,和细胞共孵育72h,弃培养液,每孔加5mg/mL的MTT溶液,放入细胞培养箱中反应4h,终止培养,弃培养液并于每孔中加入150uL DMSO溶液后,置于摇床上振荡10min。使用酶标仪在490nm处测量各孔的吸光度,计算各组药物的半数抑制浓度IC50值
结果如表1所示,游离药物FIS的IC50为31.18μM,利用脂质体负载FIS后,RMP7-EL-FIS将FIS的IC50降低至18.34μM,其中,RMP7-EL-FI-PIO组FIS的IC50值相比于RMP7-EL-FIS组降低了一半,说明RMP7-EL-FI-PIO具有更强的抑制U87细胞增殖的能力。
表现出较强的抑制肿瘤细胞增殖能力。
表1游离药物FIS、游离FIS+PIO、RMP7-EL-FIS和RMP7-EL-FI-PIO对SH-SY5Y细胞的的IC50
1.6靶向肽修饰的“杂合脂质体”纳米粒跨BBB能力的考察
Transwell小室上层加100μM胶原,风干成胶。将5×105个Bend.3细胞接种于Transwell小室微孔膜上,培养至细胞成单层生长。Transwell下层加DMEM细胞培养基以高过上层液面(具有>0.5cm的液面差)。置于细胞培养箱培养4h后,观察上下层液面差变化,没有明显变化时,说明形成了较为致密的细胞层。上层加入C6脂质体(C6-L,C6-EL,C6-RMP7-L,C6-RMP7-EL),培养4h后检测底部细胞培养液中C6脂质体的荧光强度。
结果如图5所示,RMP7-EL组的荧光信号强度最高,说明靶向肽修饰的“杂合脂质体”能够高效跨过BBB,实现脑靶向。
Claims (4)
1.一种脑靶向肽修饰的“杂合脂质体”纳米粒,其特征在于,所述“杂合脂质体”纳米粒主要由功能化脂质体和外泌体融合而成,功能化脂质体主要由靶向肽、磷脂和脂溶性活性药物构成,是通过以下方法制备而成,具体包括如下步骤:
1)制备含有磷脂、Chol、DSPE-PEG、DSPE-PEG-NHS、脂溶性活性药物的有机溶液A:
当磷脂质量为46.25mg时,有机溶剂质量以5mL计,固体质量比为磷脂:Chol:DSPE-PEG:DSPE-PEG-NHS:脂溶性活性药物=23.125:1.5:6.25:2:1.5;
2)旋转蒸发有机溶液A除去有机溶剂,PBS水化,超声破碎,过膜,得载药脂质体;
3)载药脂质体与靶向肽溶液孵育,得载药功能化脂质体:
其中,靶向肽溶液是将1mg靶向肽溶于1mL超纯水中得到,浓度为1mg/mL,靶向肽溶液与载药脂质体4℃孵育12h,所述靶向肽与DSPE-PEG-NHS的质量比=1:16;
4)提取分离获得外泌体:采用密度梯度离心法提取分离得到外泌体,转速为300g—100000g;
5)取步骤(3)所得载药功能化脂质体和步骤(4)所得外泌体挤出:其中,载药功能化脂质体以磷脂总量计,外泌体以蛋白含量计,所述载药功能化脂质体和载药外泌体质量比为5:1,使用挤出器将脂质体与外泌体挤出10-20次,收集,即得靶向肽修饰的“杂合脂质体”纳米粒。
2.如权利要求1所述的脑靶向肽修饰的“杂合脂质体”纳米粒,其特征在于,所述步骤1)中的有机溶剂为甲醇,所述磷脂选自DOPC、DOPS、DOPE、DMPC、DMPE中的一种或几种,所述脂溶性活性药物为非瑟酮,吡格列酮,尼达尼布中的一种或几种。
3.如权利要求1所述的脑靶向肽修饰的“杂合脂质体”纳米粒,其特征在于,所述步骤2)中的旋转蒸发时间为30min,水化时间为30min,超声破碎时间为5~10min。
4.如权利要求1所述的脑靶向肽修饰的“杂合脂质体”纳米粒,其特征在于,所述步骤3)中的靶向肽为杭州佳肽生物公司合成,氨基酸序列为:
H-Arg-Pro-Hyp-Gly-Thi-SerPro-4-Me-Tyr(psi CH2NH)-Arg-OH。
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