CN117257975A - 一种多功能细胞外囊泡及其制备方法和应用 - Google Patents
一种多功能细胞外囊泡及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种多功能细胞外囊泡及其制备方法和应用,属于生物医药技术领域。本发明通过将抗炎细胞外囊泡与线粒体靶向肽基药偶联,得到的多功能细胞外囊泡能够解决现有技术中细胞外囊泡功能化产品功效单一、种类不足的问题。本发明提供的多功能细胞外囊泡有着环境可塑性、能够表达靶向炎症的整合素,具有定位于炎症病变的能力,可以靶向富集到炎症部位,还具有活性氧响应功能,可定点释放药物;本发明提供的多功能细胞外囊泡还具有协同的抗炎和抗氧化的治疗作用、具有保护线粒体功能和更强的器官保护作用。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及一种多功能细胞外囊泡及其制备方法和应用。
背景技术
细胞外囊泡(extracellular vesicles, EVs)是一种由细胞释放到细胞外基质的膜性小囊泡,参与细胞通讯、细胞迁移、血管新生和肿瘤细胞生长等过程,广泛地存在于各种体液和细胞上清中,并稳定携带了一些重要的信号分子。囊泡相关功能的研究已经成为研究热点,并有望在多种疾病的早期诊断中发挥作用。
为了达到更好的功效,现有技术公开了对细胞外囊泡进行功能化改进的多种技术方案,例如中国专利CN113616674A公开了一种具有抗炎作用的细胞外囊泡,具体为M2型巨噬细胞来源的胞外囊泡,通过实验证明M2型巨噬细胞来源的EVs具有免疫调节功能,发挥抗炎及促进组织修复的作用,在急性全身性炎症反应中具有潜在的治疗作用。
但是现有技术中的细胞外囊泡功能化产品存在功效单一、种类不足的问题。
发明内容
本发明的目的在于提供一种多功能细胞外囊泡及其制备方法和应用,以解决现有技术中细胞外囊泡功能化产品功效单一、种类不足的问题。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种多功能细胞外囊泡,所述多功能细胞外囊泡的结构中包含抗炎细胞外囊泡和偶联在抗炎细胞外囊泡表面的线粒体靶向肽基药。
优选的,所述抗炎细胞外囊泡与带有活性氧响应接头的线粒体靶向肽基药通过活性氧响应的化学接头实现偶联。
优选的,所述带有活性氧响应接头的线粒体靶向肽基药为DSPE-PEG-TK-SS31。
本发明还提供了上述多功能细胞外囊泡的制备方法,包括以下步骤:
将抗炎细胞外囊泡与带有活性氧响应接头的线粒体靶向肽基药进行共孵育,在此过程中,连接基团DSPE可插入抗炎细胞外囊泡的磷脂双分子层,使得抗炎细胞外囊泡与线粒体靶向肽基药偶联,得到多功能细胞外囊泡。
优选的,所述抗炎细胞外囊泡通过包含以下步骤的制备方法得到:
将炎症因子与间充质干细胞混合,进行预刺激;
预刺激后收集条件培养基;
将条件培养基中的抗炎细胞外囊泡进行分离。
优选的,所述炎症因子包括TNF-α、INF-γ和IL-1β;
所述TNF-α的浓度为1~100ng/mL;
所述INF-γ的浓度为1~100ng/mL;
所述IL-1β的浓度为1~100ng/mL;
所述预刺激的时间为12~72h。
优选的,所述带有活性氧响应接头的线粒体靶向肽基药为DSPE-PEG-TK-线粒体靶向肽。
优选的,所述线粒体靶向肽为SS-31肽。
优选的,所述DSPE-PEG-TK-线粒体靶向肽通过包含以下步骤的制备方法得到:
将DSPE-PEG-NH2和线粒体靶向肽通过ROS响应性连接剂进行连接,得到DSPE-PEG-TK-线粒体靶向肽。
本发明还提供了上述多功能细胞外囊泡、通过上述制备方法得到的多功能细胞外囊泡在制备抗炎药物、抗氧化药物、促修复药物或防治急性肾损伤药物中的应用。
本发明的有益效果:
本发明提供的多功能细胞外囊泡具有靶向炎症功能、有着环境可塑性、能够表达靶向炎症的整合素,具有定位于炎症病变的能力,可以靶向富集到炎症部位;所述多功能细胞外囊泡还具有活性氧响应功能,可定点释放药物;本发明提供的多功能细胞外囊泡还具有协同的抗炎和抗氧化的治疗作用,具备增强的抗炎疗效,可以减少炎症细胞浸润和炎症因子表达,发挥抗炎作用;还具有保护线粒体功能,发挥减轻氧化应激损伤作用,协同促进组织器官修复;另外具有增强的器官保护作用,细胞外囊泡与单独原料组分相比,进一步提高了肾功能、改善了肾脏病理损伤,发挥更优的保护作用。
附图说明
图1为三种细胞外囊泡合成图;
图2为三种细胞外囊泡微观形态图,其中A为细胞外囊泡(EV)电镜图;B为抗炎的细胞外囊泡(pEV)电镜图、C为多功能细胞外囊泡(pEV-TK-SS31)电镜图;
图3为三种细胞外囊泡粒径分布图,其中A为细胞外囊泡(EV)的粒径分析图;B为抗炎的细胞外囊泡(pEV)的粒径分析图;C为多功能细胞外囊泡(pEV-TK-SS31)的粒径分析图;
图4为动物肾脏内细胞外囊泡荧光成像图;
图5为细胞外囊泡荧光信号统计图;
图6为两种细胞外囊泡蛋白质组学检测图,其中A为抗炎细胞外囊泡(pEV)和细胞外囊泡(EV)做蛋白质组学的火山图;B为抗炎细胞外囊泡(pEV)和细胞外囊泡(EV)做蛋白质组学的差异蛋白热图;C为差异富集通路统计结果图;
图7为具有靶向和抗炎抗氧化功能的细胞外囊泡炎症富集图,其中A为免疫蛋白印迹结果,表明多功能细胞外囊泡高表达Integrin α4β1,B为粘附蛋白Integrin α4和Integrin β1组成的二聚体Integrin α4β1对炎症部位高表达的ICAM-1有高亲和性示意图;
图8为具有靶向和抗炎抗氧化功能的细胞外囊泡的活性氧响应性药物释放图;
图9为免疫荧光追踪共定位图及共定位分析图,其中A为免疫荧光追踪共定位图,B为信号共定位分析图;
图10为动物肾脏病理和肾脏功能指标图,其中A为周期性酸-希夫(PAS)染色结果图;B为急性肾小管坏死(ATN)评分分析结果图;C为血肌酐(SCr)评估结果图;D为血尿素氮(BUN)评估结果图;
图11为细胞外囊泡发挥抗炎和抗氧化协同作用图,其中A为巨噬细胞浸润的免疫荧光检测结果图;B为不同实验组对中性粒细胞浸润免疫荧光结果检测图;C为氧化应激损伤的标志物亚硝基酪氨酸(nitrotyrosine)的免疫荧光检测结果图;
图12为氧化应激损伤的标志物丙二醛(MDA)指标图。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
本发明提供了一种多功能细胞外囊泡,所述多功能细胞外囊泡的结构中包含抗炎细胞外囊泡和偶联在抗炎细胞外囊泡表面的线粒体靶向肽基药,所述抗炎细胞外囊泡与线粒体靶向肽基药优选通过活性氧响应的化学接头实现偶联,所述线粒体靶向肽基药优选为SS31。
本发明还提供了上述多功能细胞外囊泡的制备方法,包括以下步骤:
将抗炎细胞外囊泡与带有活性氧响应接头的线粒体靶向肽基药进行共孵育,连接基团DSPE可插入抗炎细胞外囊泡的磷脂双分子层,使得抗炎细胞外囊泡与线粒体靶向肽基药偶联,得到功能化细胞外囊泡,当采用DSPE-PEG-TK-SS31时,其与抗炎细胞外囊泡共孵育过程中DSPE通过疏水作用插入细胞外囊泡的磷脂双分子层,制备的功能化细胞外囊泡具有协同抗炎和抗氧化的治疗作用,促进组织器官修复。
所述抗炎细胞外囊泡优选通过包含以下步骤的制备方法得到:将炎症因子与间充质干细胞混合,进行预刺激;预刺激后收集条件培养基;将条件培养基中的抗炎细胞外囊泡进行分离,所述炎症因子优选包括TNF-α、INF-γ和IL-1β,所述TNF-α的浓度优选为1~100ng/mL;所述INF-γ的浓度优选为1~100ng/mL;所述IL-1β的浓度优选为1~100ng/mL;所述预刺激的时间优选为12~72h;所述预刺激的步骤是基于间充质干细胞的抗炎能力的可塑性具备的环境依赖特征,通过模拟急性肾损伤炎性微环境的策略“教育”间充质干细胞,进而制备得到抗炎细胞外囊泡。
在本发明中,所述带有活性氧响应接头的线粒体靶向肽基药优选为DSPE-PEG-TK-线粒体靶向肽,所述线粒体靶向肽优选为SS-31肽;
所述DSPE-PEG-TK-线粒体靶向肽优选通过包含以下步骤的制备方法得到:将DSPE-PEG-NH2和线粒体靶向肽通过ROS响应性连接剂进行连接,得到DSPE-PEG-TK-线粒体靶向肽。
本发明还提供了上述多功能细胞外囊泡、通过上述制备方法得到的多功能细胞外囊泡在制备抗炎药物、抗氧化药物、促修复药物或防治急性肾损伤药物中的应用;所得的细胞外囊泡具有利用组分之一的抗炎细胞外囊泡定位炎症病变的能力,可以靶向富集到炎症部位;细胞外囊泡的活性氧接头在组织损伤中被高表达的活性氧切割,可定点释放药物,协同抗炎和抗氧化的治疗作用以促进组织器官修复。
在本发明中,所述间充质干细胞与炎症因子混合之前优选包括以下步骤:使用培养基体外培养间充质干细胞,待间充质干细胞达到40%融合度时使用炎症因子预刺激间充质干细胞46~50h;然后利用pH=7.4的磷酸盐缓冲溶液清洗细胞两次并去除胎牛血清,收集条件培养基。
优选的,培养基为Dulbecco改良的Eagle's培养基,其含10%的胎牛血清和100U/mL的青霉素-链霉素;炎症因子包括1~100ng/mL的TNF-α、1~100ng/mL的INF-γ和1~100ng/mL的IL-1β;预刺激时间为48h。
优选的,去除胎牛血清的方法为:用去除细胞外囊泡的胎牛血清替换胎牛血清或者超速离心胎牛血清。
优选的,所述将条件培养基中的抗炎细胞外囊泡进行分离的步骤优选包括以下步骤:
将条件培养基在3~5℃及450~550×g条件下第一次离心8~12min;再在3~5℃及1500~2500×g条件下第二次离心15~25min;再在3~5℃及8000~12000×g条件下第三次离心25~35min;用0.22μm过滤器过滤后在3~5℃及80000~120000×g条件下第四次离心65~75min;弃掉上清液,用磷酸盐缓冲溶液重悬,再以80000~120000×g第五次离心1.5~2.5h;去除上清液后重悬于磷酸盐缓冲溶液中,并于-80℃温度下储存得抗炎细胞外囊泡。
优选的,第一次离心条件为4℃、500×g及10min;第二次离心条件为4℃、2000×g及20min;第三次离心条件为4℃、10000×g及30min;第四次离心条件为4℃、100000×g及70min;第五次离心条件为100000×g及2h。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
1)使用含10%胎牛血清和100U/mL青霉素-链霉素的Dulbecco改良的Eagle's培养基体外培养间充质干细胞,待间充质干细胞达到40%融合度时,使用包含50ng/mL的TNF-α、50ng/mL的INF-γ和50ng/mL的IL-1β的炎症因子预刺激间充质干细胞48h;然后利用pH=7.4磷酸盐缓冲溶液清洗细胞两次,将普通10%胎牛血清换用商售的10%去除细胞外囊泡的胎牛血清,或者通过将普通胎牛血清超速离心(100000~120000×g,12h)获得去除细胞外囊泡的胎牛血清,再收集条件培养基;
2)将条件培养基在4℃及500×g条件下第一次离心10min;再在4℃及2000×g条件下第二次离心20min;再在4℃及10000×g条件下第三次离心30min;用0.22μm过滤器过滤;在4℃及100000×g条件下第四次离心70min;弃掉上清液,并用磷酸盐缓冲溶液重悬后,再以100000×g第五次离心2h;去除上清液后重悬于磷酸盐缓冲溶液中,用BCA试剂盒检测浓度,并于-80℃温度下储存得抗炎细胞外囊泡;
3)首先将羧基末端的酮缩硫醇(thioketal,TK)与DSPE-PEG2000-NH2通过氨基与羧基的缩合反应相连接,所得反应液用去离子水透析(MWCO,1000Da)纯化2天,然后冻干,制得DSPE-PEG2000-TK,所述TK与DSPE-PEG2000-NH2的投料摩尔比为1~100 : 1。随后将DSPE-PEG2000-TK和SS31进一步通过氨基与羧基的缩合反应相连接,所得反应液用去离子水透析(MWCO,1000Da)纯化2天,然后冻干,制得DSPE-PEG2000-TK-NHS,所述TK与DSPE-PEG2000-TK和SS-31的投料摩尔比为1 : 0.1~100。最后,将DSPE-PEG-TK-SS31与抗炎细胞外囊泡共孵育(4℃,6~24h或常温0.3~2h),DSPE通过疏水作用插入细胞外囊泡的磷脂双分子层,得到具有靶向和抗炎抗氧化功能的细胞外囊泡pEV-TK-SS31。
实施例2
1)使用含10%胎牛血清和100U/mL青霉素-链霉素的Dulbecco改良的Eagle's培养基体外培养间充质干细胞,待间充质干细胞达到40%融合度时,使用包含1ng/mL的TNF-α、1ng/mL的INF-γ和1ng/mL的IL-1β的炎症因子预刺激间充质干细胞46h;然后利用pH=7.4磷酸盐缓冲溶液清洗细胞两次,超速离心去除胎牛血清,再收集条件培养基;
2)将条件培养基在3℃及450×g条件下第一次离心12min;再在3℃及1500×g条件下第二次离心25min;再在3℃及8000×g条件下第三次离心35min;用0.22μm过滤器过滤;在3℃及80000×g条件下第四次离心75min;弃掉上清液,并用磷酸盐缓冲溶液重悬后,再以80000×g第五次离心2.5h;去除上清液后重悬于磷酸盐缓冲溶液中,用BCA试剂盒检测浓度,并于-80℃温度下储存得抗炎细胞外囊泡;
3)将酮缩硫醇分别与氨基末端的DSPE-PEG-NH2和含氨基的SS31多肽反应,得到具有ROS响应性的DSPE-PEG-TK-SS31,再将DSPE-PEG-TK-SS31与抗炎细胞外囊泡共孵育,DSPE通过疏水作用插入细胞外囊泡的磷脂双分子层,得到具有靶向和抗炎抗氧化功能的细胞外囊泡pEV-TK-SS31,此步骤与实施例1相同。
实施例3
一种具有靶向和抗炎抗氧化功能的细胞外囊泡,其经过以下步骤制得:
1)使用含10%去除细胞外囊泡的胎牛血清和100U/mL青霉素-链霉素的Dulbecco改良的Eagle's培养基体外培养间充质干细胞,待间充质干细胞达到40%融合度时,使用包含100ng/mL的TNF-α、100ng/mL的INF-γ和100ng/mL的IL-1β的炎症因子预刺激间充质干细胞50h;然后利用pH=7.4磷酸盐缓冲溶液清洗细胞两次,再收集条件培养基;
2)将条件培养基在5℃及550×g条件下第一次离心8min;再在5℃及2500×g条件下第二次离心15min;再在5℃及12000×g条件下第三次离心25min;用0.22μm过滤器过滤;在5℃及120000×g条件下第四次离心65min;弃掉上清液,并用磷酸盐缓冲溶液重悬后,再以120000×g第五次离心1.5h;去除上清液后重悬于磷酸盐缓冲溶液中,用BCA试剂盒检测浓度,并于-80℃温度下储存得抗炎细胞外囊泡;
3)将酮缩硫醇分别与氨基末端的DSPE-PEG-NH2和含氨基的SS31多肽反应,得到具有ROS响应性的DSPE-PEG-TK-SS31,再将DSPE-PEG-TK-SS31与抗炎细胞外囊泡共孵育,DSPE通过疏水作用插入细胞外囊泡的磷脂双分子层,得到具有靶向和抗炎抗氧化功能的细胞外囊泡pEV-TK-SS31,此步骤与实施例1相同。
对比例1
一种细胞外囊泡(EV),与实施例1相比,该细胞外囊泡未经过炎症教育策略培养和未连接线粒体靶向肽基药。
对比例2
一种抗炎细胞外囊泡(pEV),与实施例1相比,该细胞外囊泡未连接线粒体靶向肽基药,其余步骤与实施例1相同。
对比例3
一种抗炎细胞外囊泡(EV-TK-SS31),与实施例1相比,该细胞外囊泡未经过炎症教育策略培养,其余步骤与实施例1相同。
实验例
1、细胞外囊泡的合成与表征。对对比例1、对比例2以及实施例1的制备流程、微观形态以及粒径分布进行表征,结果分别如图1~3所示。
结果显示,工程化修饰后的细胞外囊泡仍然保持着细胞外囊泡的标志蛋白、粒径分布等性质。
2、细胞外囊泡的靶向性实验。体内分布实验,利用DiD标记分别标记对比例1及实施例1制备的细胞外囊泡,构建实施例1工程化细胞外囊泡pEV-TK-SS31,同时将对比例3工程化细胞外囊泡EV-TK-SS31作为对照;建立急性肾损伤模型后将两者进行尾静脉注射,于注射24h后处死动物,留取肾脏进行荧光成像。
结果如图4及图5所示,由实施例1抗炎细胞外囊泡组装的pEV-TK-SS31相对更多地积蓄在炎症肾脏,提示pEV-TK-SS31有炎症靶向性。
细胞外囊泡的膜蛋白介导其内吞摄取,有多项研究表明细胞外囊泡可以通过表面的粘附分子靶向到炎症部位。
因此,本发明将实施例1所制备的细胞外囊泡(pEV-TK-SS31)中决定与细胞的相互作用的组分-抗炎细胞外囊泡(pEV),和对比例1细胞外囊泡(EV)进行蛋白质组学检测聚焦关注粘附蛋白的差异表达。
结果如图6所示,蛋白质组学结果表明包括整合素在内的粘附蛋白是pEV的差异富集通路。如图7所示,pEV含有丰富的粘附蛋白Integrin α4和Integrin β1,Western blot验证了上述两个整合素蛋白的高表达。粘附蛋白Integrin α4和Integrin β1组成的二聚体Integrin α4β1对炎症部位高表达的ICAM-1具有高亲和性,因此表明富含Integrin α4β1的工程化细胞外囊泡pEV-TK-SS31可以靶向肾脏炎症区域,在肾脏炎症部位富集。
3、细胞外囊泡的活性氧响应性能实验。在实施例1细胞外囊泡pEV-TK-SS31中引入了对活性氧反应的结构(thioketal linker,TK linker),TK linker可在病变中被活性氧裂解以实现药物的按需释放(图8)。为了更好地证明TK连接体对活性氧反应性释放药物的能力,还合成了一个细胞外囊泡的类似物pEV-AA-SS31,即将TK linker替换成对活性氧不敏感的AA linker。评估了上述两者在细胞内的可控药物释放,pEV被phk26标记为红色,SS31被FITC替代用以可视化,利用免疫荧光以追踪二者在细胞内的定位。人肾小管上皮细胞(HK2 cells)利用或不利于活性氧刺激剂过氧化氢(H2O2)处理,同时用细胞外囊泡pEV-TK-FITC或pEV-AA-FITC治疗。
结果如图9所示,没有H2O2刺激时,无论是被TK linker还是被AA linker连接的pEV和FITC的信号都很好的重叠在一起,而存在H2O2损伤后,对活性氧不敏感的AA linker连接的pEV和FITC二者信号仍共定位,而被TK linker连接的pEV和FITC的信号不重叠,二者不存在共定位,表明TK linker被ROS裂解后pEV和FITC被释放,证实了TK linker的活性氧响应性。
4、细胞外囊泡对急性肾损伤的疗效实验。为了评估pEV-TK-SS31的治疗效果,建立了缺血/再灌注(I/R)诱导的急性肾损伤(I/R-AKI);组织病理学损伤通过周期性酸-希夫(PAS)染色进行评估。坏死以及死细胞脱落到肾小管管腔或变性蛋白质沉淀而形成的管型是肾脏损伤的重要标志(图10-A)。与假手术动物(Sham)相比,I/R组的广泛坏死(箭头所示)和管型形成(星号所示)都相当严重;经SS31、pEV或pEV-AA-SS31处理后,局灶性坏死和管型形成依然存在,而pEV-TK-SS31处理后,肾脏损伤明显减轻,坏死和管型形成更少,而且正常肾脏结构保存完好,近端肾小管刷状缘与假手术组的肾脏结构组相似。同样,急性肾小管坏死(ATN)评分分析也量化了肾小管损伤,并与上述观察结果一致(图10-B)。同时,还评估了肾功能的重要临床指标--血清肌酐(SCr)和血尿素氮(BUN)(图10-C及图10-D)。从SCr和BUN的增加可以看出,受到I/R损伤的小鼠出现了严重的肾功能障碍。相比之下,SS31、pEV或pEV-AA-SS31治疗可在一定程度上部分挽救肾功能,而pEV-TK-SS31治疗的效果更好。这些结果表明pEV-TK-SS31能在功能和组织病理学水平上更有效地减轻I/R引起的急性肾损伤。
5、细胞外囊泡的抗炎和抗氧化的疗效实验。检测pEV-TK-SS31对炎症和氧化应激的改善的作用,分别利用免疫荧光检测了急性肾损伤中炎症最常见的巨噬细胞(图11-A),结果表明,急性肾损伤(IR)组红色荧光标记的巨噬细胞相比假手术组明显增多,三个治疗组pEV、SS31和pEV-AA-SS31均对巨噬细胞浸润有一定缓解,而pEV-TK-SS31对减少巨噬细胞浸润疗效最好。类似的,pEV-TK-SS31也显示了对中性粒细胞浸润(图11-B)的改善,提示pEV-TK-SS31对炎症具有良好的治疗作用。
检测氧化应激损伤,亚硝基酪氨酸(nitrotyrosine)的产生代表氧化应激蛋白损伤,结果显示,单独使用SS31或pEV-AA-SS31可部分减少亚硝基酪氨酸的表达,而pEV-TK-SS31则几乎能完全消除亚硝基酪氨酸(图11-C)。与亚硝基酪氨酸减少同时出现的是脂质过氧化程度和细胞氧化损伤的另一个指标--MDA,pEV-TK-SS31也能很好地减少MDA的表达(图12)。上述结果证实,细胞外囊泡能发挥抗炎和抗氧化的协同作用。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种多功能细胞外囊泡,其特征在于,所述多功能细胞外囊泡的结构中包含抗炎细胞外囊泡和偶联在抗炎细胞外囊泡表面的线粒体靶向肽基药。
2.根据权利要求1所述的多功能细胞外囊泡,其特征在于,所述抗炎细胞外囊泡与带有活性氧响应接头的线粒体靶向肽基药通过活性氧响应的化学接头实现偶联。
3.根据权利要求1或2所述的多功能细胞外囊泡,其特征在于,所述线粒体靶向肽基药为带有活性氧响应接头的DSPE-PEG-TK-SS31。
4.权利要求1~3任一项所述的多功能细胞外囊泡的制备方法,其特征在于,包括以下步骤:
将抗炎细胞外囊泡与带有活性氧响应接头的线粒体靶向肽基药进行共孵育,使抗炎细胞外囊泡与线粒体靶向肽基药偶联,得到多功能细胞外囊泡。
5.根据权利要求4所述的多功能细胞外囊泡的制备方法,其特征在于,所述抗炎细胞外囊泡通过包含以下步骤的制备方法得到:
将炎症因子与间充质干细胞混合,进行预刺激;
预刺激后收集条件培养基;
将条件培养基中的抗炎细胞外囊泡进行分离。
6.根据权利要求5所述的多功能细胞外囊泡的制备方法,其特征在于,所述炎症因子包括TNF-α、INF-γ和IL-1β;
所述TNF-α的浓度为1~100ng/mL;
所述INF-γ的浓度为1~100ng/mL;
所述IL-1β的浓度为1~100ng/mL;
所述预刺激的时间为12~72h。
7.根据权利要求4所述的多功能细胞外囊泡的制备方法,其特征在于,所述带有活性氧响应接头的线粒体靶向肽基药为DSPE-PEG-TK-线粒体靶向肽。
8.根据权利要求7所述的多功能细胞外囊泡的制备方法,其特征在于,所述线粒体靶向肽为SS-31肽。
9.根据权利要求7所述的多功能细胞外囊泡的制备方法,其特征在于,所述DSPE-PEG-TK-线粒体靶向肽通过包含以下步骤的制备方法得到:
将DSPE-PEG-NH2和线粒体靶向肽通过ROS响应性连接剂进行连接,得到DSPE-PEG-TK-线粒体靶向肽。
10.权利要求1~3任一项所述的多功能细胞外囊泡、通过权利要求4~9任一项所述的制备方法得到的多功能细胞外囊泡在制备抗炎药物、抗氧化药物、促修复药物或防治急性肾损伤药物中的应用。
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