CN116159036A - 一种细胞外囊泡载药***及其制备方法和应用 - Google Patents
一种细胞外囊泡载药***及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及生物医学技术领域,具体涉及一种细胞外囊泡载药***及其制备方法和应用。所述细胞外囊泡载药***包括细胞外囊泡以及负载于细胞外囊泡内的CircRNA和抗结核药物。本发明以细胞外囊泡作为CircRNA和抗结核药物的共载体,高效装载抗结核免疫调控功能CircRNA和抗结核药物,再通过对细胞外囊泡进行表面靶向修饰能够进一步增强该细胞外囊泡载药***的靶向性。该细胞外囊泡载药***通过结合CircRNA的抗结核免疫调控功能和抗结核药物的结核分歧杆菌抑制作用,更加有效杀伤和清除宿主细胞内的结核分歧杆菌,从而有望为安全高效的抗结核药物开发提供新策略。
Description
技术领域
本发明涉及生物医学技术领域,具体涉及一种细胞外囊泡载药***及其制备方法和应用。
背景技术
目前的药物传递***(drug delivery system),如纳米给药***,通常使用各种稳定剂制备稳定的纳米***,装载药物后,可以主动或被动地聚集到靶器官、细胞或细胞器,增强药物的靶向性;利用纳米颗粒装载药物还可以提高药物的稳定性和可溶性,促进药物的跨膜转运,并延长药物的循环时间以提高药物药效,并降低游离药物对非靶器官/细胞的毒性。
纳米医学可以促进药物的临床转化并改善患者治疗时的副反应,但目前只有少数聚合纳米药物得到了FDA的批准并用于临床。目前已获批准的纳米药物,很少被推荐作为一线治疗药物,而且部分药物只在一小部分患者中显示出更好的疗效。尽管现有的工程化纳米技术能突破传统载药技术的限制,显示出增强药物疗效的潜力。但也存在一些不足,例如脂质体载药的药物易泄漏、稳定性较差;无机纳米颗粒常使用重金属制剂,溶解度低、毒副作用风险高。因此,开发安全性高、靶向性好的纳米***作为递送载体仍是生物医学领域的重大科学难题。
细胞外囊泡(extracellular vesicles,EVs)是细胞来源的纳米级囊泡,主要包括外泌体和微囊泡等。在不同类型的内源性或外源性刺激作用下,细胞质膜可以脱落产生或由核内体分泌出不同大小的囊泡,100-1000nm称为微囊泡,而30-150nm称为外泌体。细胞外囊泡分布在生物体液中,能携带核酸、蛋白质和脂质等生物分子在体液中循环,并且可以通过血脑屏障,在细胞间通讯中发挥重要作用;同时,细胞外囊泡具有的典型的脂质双分子层结构,能够保护核酸不被降解,是基因治疗中核酸(如siRNA和miRNAs)的理想载体,因此具有作为生物分子传递载体的潜能。此外,细胞外囊泡被释放到细胞外环境后,可被不同类型的细胞摄取,主要通过两种方式进行细胞信号转导和交流:一是与特定细胞膜分子的结合;二是通过内吞作用(如网格蛋白/小窝蛋白介导的内吞作用、巨胞饮作用、吞噬作用、脂筏介导的摄取等)进入靶细胞。进入细胞后,细胞外囊泡可以将其装载的货物释放到目标细胞中并执行其生物功能。由于细胞外囊泡在细胞脱落过程中携带了来源细胞的磷脂双分子层结构,可以更有效地与受体细胞融合;此外,通过对细胞外囊泡表面进行修饰还可以实现更高效的靶向作用,使它们能够将药物、蛋白质等生物分子传递到目标组织。
综上所述,细胞外囊泡是理想的药物和生物分子递送载体。与脂质体或其他纳米颗粒等合成载体相比,细胞外囊泡作为细胞的产物,更具有良好的生物相容性、低免疫原性和跨越血脑屏障等生物屏障的能力。细胞外囊泡作为各种疾病治疗的纳米载体具有很大的前景,有利于提高临床疗效和安全性。
细胞RNA在生理和病理环境中发挥重要的调控作用,因此,探寻各种疾病状态下的特异性RNA表达谱并探究其致病机制,有望将特异性RNA作为疾病的候选生物标志物和潜在的治疗靶点。除mRNA外,许多非编码RNA(ncRNAs)或环状RNA(CircRNAs)协同控制基因表达,构成了药物开发的多样潜在靶点。目前用作治疗药物的RNA分子或类似物,主要利用化学合成的单链寡核苷酸,通过与靶向mRNA碱基对互补,阻断其作用并抑制基因表达;或者利用载体传递miRNAs,增强疾病相关缺陷的miRNAs的表达等。但RNA药物容易在血液中降解,且需通过细胞膜屏障才能到达细胞内靶点;此外,RNA药物通常需要载体传递,且使用广泛的化学修饰以提高RNA稳定性,但也面临可诱导免疫原性反应的风险,临床应用受限。研究表明,在活细胞中制备天然的RNA药物开发有望将诱导免疫反应的风险降到最低。因此,利用细胞外囊泡作为RNA和药物共载体,能实现药物稳定性和组织特异性靶向,避免或最小化脱靶效应和免疫原性,有利于提高临床疗效和安全性。
研究表明,特定RNA如miRNAs、CircRNAs,能被选择性富集到细胞外囊泡中。作为一种由真核转录组表达的内源性、组织特异性RNA,CircRNAs不具有5’末端帽子和3’末端poly(A)尾,是通过外显子或内含子环化,将3’和5’末端连接起来形成的共价环状结构,能抵抗RNase R消化。通过高通量RNA测序和微阵列分析研究,不同来源的细胞外囊泡中含有的不同CircRNAs能被识别,且研究表明细胞外囊泡中的CircRNAs能在受体细胞中发挥细胞外功能和关键作用。而基于其较其他种类RNA更稳定的优势,CircRNAs也因此更有望被用于生物医学领域。
基于此,本发明提出了采用细胞外囊泡作为CircRNA和抗结核药物的共载体,进而形成细胞外囊泡载药***,为制备抗结核药物或抗结核治疗提供新策略。
发明内容
本发明的目的是为了解决上述现有技术的不足而提供一种细胞外囊泡载药***及其制备方法和应用。
本发明的目的通过下述技术方案予以实现:本发明提供了一种细胞外囊泡载药***,包括细胞外囊泡以及负载于细胞外囊泡内的CircRNA和抗结核药物。
进一步的,所述抗结核药物为利福平。
进一步的,所述细胞外囊泡为外泌体或微囊泡。
进一步的,所述细胞外囊泡为经表面靶向修饰的外泌体。本发明中利用外泌体膜结构磷脂天然交换的特性,用DSPE-PEG-MAN对外泌体进行表面靶向修饰,进而增强该细胞外囊泡载药***的靶向性。
进一步的,所述CircRNA为circTRAPPC6B。
在本发明中,通过利用高表达特异性CircRNA的细胞外囊泡,并对其进行抗结核药物的装载,使该细胞外囊泡作为CircRNA和抗结核药物的共载体,进而形成所述细胞外囊泡载药***;再者,为了增强细胞外囊泡的***靶向性,进一步对该细胞外囊泡进行表面靶向修饰。本发明所述细胞外囊泡载药***通过结合宿主导向治疗和药物治疗的优势,有望为新型抗结核药物开发或抗结核病治疗提供新策略和新方法。
本发明提供了一种细胞外囊泡载药***的制备方法,包括如下步骤:
(1)提取高表达CircRNA外泌体:
(2)采用单纯孵育法或电穿孔法将抗结核药物装载到步骤(1)制备的高表达CircRNA外泌体中;
(3)利用超滤管洗去游离的抗结核药物,加入PBS洗涤,多次离心直至完全去除游离的抗结核药物,再用PBS重悬超滤管收集得到CircRNA和抗结核药物共负载外泌体;
(4)对步骤(3)获得CircRNA和抗结核药物共负载外泌体进行表面靶向修饰,即得到所述细胞外囊泡载药***。
进一步的,在步骤(2)中,所述抗结核药物为利福平,所述电穿孔法的具体步骤为:取100μL的步骤(1)制备浓度为1mg/mL的高表达CircRNA外泌体于EP管中,加入100μL的浓度为2mg/mL的利福平,颠倒混匀置于冰上,设置电穿孔仪器的操作条件为1000V、10ms和2pulses,将200μL的高表达CircRNA外泌体和利福平混合物进行电穿孔,并收集电穿孔后的外泌体,置于37℃孵育30min以恢复外泌体膜结构。
进一步的,在步骤(4)中,表面靶向修饰的具体操作为:取1mL的步骤(3)制备的浓度为500μg/mL的CircRNA和抗结核药物共负载外泌体,加入1mL的浓度为20μg/mL的DSPE-PEG-MAN,颠倒混匀,置于4℃摇床避光孵育过夜;再利用超滤管将游离的DSPE-PEG-MAN洗去,加入PBS洗涤,重复离心多次。
本发明还提供了上述细胞外囊泡载药***在抗结核药物的应用。
本发明的有益效果在于:本发明提出了一种细胞外囊泡载药***及其制备方法,其中以细胞外囊泡作为CircRNA和抗结核药物的共载体,细胞外囊泡具有良好的生物相容性,并且能够高效装载抗结核免疫调控功能CircRNA和抗结核药物,再通过对细胞外囊泡进行表面靶向修饰能够进一步增强该细胞外囊泡载药***的靶向性。该细胞外囊泡载药***通过结合CircRNA的抗结核免疫调控功能和抗结核药物的结核分歧杆菌抑制作用,更加有效杀伤和清除宿主细胞内的结核分歧杆菌,从而有望为安全高效的抗结核药物开发提供新策略。
附图说明
图1是荧光显微镜下观察正常未转染细胞和转染高表达CircRNA慢病毒细胞的荧光表达情况;
图2是RT-qPCR分别检测正常未转染细胞和转染高表达circTRAPPC6B慢病毒细胞中circTRAPPC6B的表达水平;
图3是Western blot检测正常未转染细胞和转染高表达CircRNA慢病毒细胞分泌的外泌体中阳性蛋白(ALIX、CD63、TSG101)和阴性蛋白(GRP94)的表达情况;
图4是粒径分析仪检测正常未转染细胞和转染高表达CircRNA慢病毒细胞分泌的外泌体的粒径大小;
图5是紫外分光光度法(480nm)检测单纯孵育法和电穿孔法制备的CircRNA和抗结核药物共负载外泌体中的药物浓度(n=5);
图6是RT-qPCR检测正常未转染细胞和转染高表达circTRAPPC6B慢病毒细胞分泌的外泌体及经表面靶向修饰的CircRNA和抗结核药物共负载外泌体中circTRAPPC6B的表达水平;
图7是Western blot检测单纯负载CircRNA外泌体、经表面靶向修饰的CircRNA和抗结核药物共负载外泌体中阳性蛋白(ALIX、CD63、TSG101)和阴性蛋白(GRP94)的表达情况;
图8是粒径分析仪检测单纯负载CircRNA外泌体、经表面靶向修饰的CircRNA和抗结核药物共负载外泌体的粒径大小;
图9是透射电镜观察单纯负载CircRNA外泌体、经表面靶向修饰的CircRNA和抗结核药物共负载外泌体的形态(放大倍数为40000),**P<0.01,***P<0.001;
图10是流式细胞术检测巨噬细胞与两种染色外泌体共孵育一定时间后,细胞内的荧光强度变化;
图11是巨噬细胞内的荧光值变化情况分析,***P<0.001;
图12是粒径分析正常未转染细胞和转染高表达circTRAPPC6B慢病毒细胞产生的两种微囊泡的粒径大小;
图13是RT-qPCR检测两种微囊泡中circTRAPPC6B的表达水平;
图14是紫外分光光度法(480nm)检测微囊泡中的药物浓度(n=5),*P<0.05。
具体实施方式
为了便于本领域技术人员的理解,下面结合实施例对本发明作进一步的说明,实施方式提及的内容并非对本发明的限定。
实施例1
提取高表达CircRNA外泌体的具体操作为:(1)通过设计和构建特异性CircRNA(以具有抗结核免疫调控功能的CircRNA—circTRAPPC6B为例)的高表达慢病毒,利用慢病毒转染293T细胞。取24孔板每孔计数1×105个细胞铺板,培养24h后以换液的方式加入500μL含2×106TU的慢病毒(MOI=10)的含血清培养基(取10μL慢病毒(1×108TU/mL)加入到490μL的含血清培养基中),继续培养48h。转染48h后在荧光显微镜下可见明显的绿色荧光表达,继续培养3-4天,用胰酶消化、收集细胞,1000rpm离心5min,弃上清,用1mL的PBS重悬沉淀,重复离心2次,最后用500μL的PBS重悬细胞沉淀,收集到流式管中。通过流式分选仪器,分选出所有含绿色荧光的细胞,分选效率为89%,从而筛选出所有含绿色荧光的CircRNA稳定高表达的细胞系。
(2)将CircRNA稳定高表达的细胞系在37℃、含5%CO2的环境下培养一段时间,收集48h内的细胞培养上清。通过4℃300×g离心10min,收集离心后上清;4℃2000×g离心10min,收集离心后上清;4℃10000×g离心10min,收集离心后上清,进行0.22μm过滤器过滤,将以上过滤后细胞上清加入100KD超滤管进行超滤,收集超滤后的浓缩液,加入外泌体沉淀试剂盒试剂Exoquick-TC(浓缩液与Exoquick-TC的体积比例为5:1),摇匀放置4℃沉淀24h,通过3000×g离心10min,弃上清;3000×g离心5min,弃去残余上清,用PBS洗涤管壁3次,弃去上清,再加入500μL的PBS重悬沉淀,即获得高表达CircRNA外泌体,于-80℃保存便于下述实验。
在本实施例中,以正常未转染细胞为正常对照组,采用荧光显微镜观察分选后的细胞荧光表达情况,其结果显示,与正常未转染细胞(NC-cells)相比,成功转染的细胞(OE-cells)稳定表达绿色荧光蛋白,且荧光强度高(图1)。通过RT-qPCR检测正常未转染细胞(NC-cells)和CircRNA稳定转染细胞(OE-cells)中CircRNA的表达水平,结果显示,CircRNA稳定转染细胞的CircRNA表达水平显著高于正常对照组(图2)。以上结果表明本实施例已成功构建CircRNA稳定高表达的细胞系。
再者,通过Western blot检测高表达CircRNA外泌体中阳性蛋白ALIX、CD63、TSG101和阴性蛋白GRP94的表达情况(图3),结果显示正常未转染细胞分泌的外泌体(Exo)和CircRNA稳定转染细胞分泌的外泌体(CircRNA@Exo)均能高表达阳性蛋白,而阴性蛋白不表达,进而能够确认前述两种外泌体的纯度。通过粒径分析仪检测前述两种外泌体的粒径大小(图4),结果显示,前述两种外泌体粒径大小均分布在60-120nm内。以上方法共同确认通过本实施例上述制备步骤能够成功提取稳定高表达CircRNA外泌体。
实施例2
本实施例提供了一种细胞外囊泡载药***(经表面靶向修饰的CircRNA和抗结核药物共负载外泌体)的制备方法包括如下步骤:
(1)按照实施例1的操作步骤提取高表达CircRNA外泌体;
(2)分别采用单纯孵育法和电穿孔法两种方式将利福平装载到步骤(1)所获得高表达CircRNA外泌体中,其中单独孵育的具体操作为:取100μL的实施例1所获得高表达CircRNA外泌体(1mg/mL)于EP管中,加入100μL的利福平(2mg/mL),颠倒混匀置于37℃孵育30min。
电穿孔法的具体操作为:取100μL的实施例1所获得高表达CircRNA外泌体(1mg/mL)于EP管中,加入100μL的利福平(2mg/mL),颠倒混匀置于冰上,利用电穿孔仪器(1000V,10ms,2pulses)将200μL的高表达CircRNA外泌体和利福平混合物进行电穿孔,收集电穿孔后的外泌体,置于37℃孵育30min以恢复外泌体膜结构。
(3)利用100KD超滤管分别将步骤(2)中单纯孵育法和电穿孔法收集的外泌体中游离的利福平洗去,每次3000×g离心15min,加入PBS洗涤,重复离心6次,通过紫外分光光度法检测最后一次洗涤的流穿液中是否含有利福平(吸光度为480nm),直至确认利福平洗涤完全。取500μL的PBS重悬超滤管中收集到的外泌体,即获得CircRNA和抗结核药物共负载外泌体。
(4)利用外泌体膜结构磷脂天然交换的特性,用DSPE-PEG-MAN(1mL,20μg/mL)对步骤(3)获得CircRNA和抗结核药物共负载外泌体(1mL,500μg/mL)进行表面靶向修饰,其具体操作为:取1mL的步骤(3)获得CircRNA和抗结核药物共负载外泌体(500μg/mL),加入1mL的DSPE-PEG-MAN(20μg/mL),颠倒混匀,置于4℃摇床避光孵育过夜。利用100KD超滤管将游离的DSPE-PEG-MAN洗去,每次3000×g离心15min,加入PBS洗涤,重复离心3次,即获得经表面靶向修饰的CircRNA和抗结核药物共负载外泌体(Man-CircRNA@Rif@Exo),置于-80℃避光保存。
在本实施例中,以正常未转染细胞为正常对照组,通过紫外分光光度法(480nm)分别检测单纯孵育法(Incubation)和电穿孔法(Electronporation)制备得到的CircRNA和抗结核药物共负载外泌体的药物浓度(图5),结果显示单纯孵育法和电穿孔法都能使外泌体成功装载抗结核药物(利福平),但与单纯孵育法相比,可以确认的是电穿孔法使外泌体装载抗结核药物的效率更高。通过RT-qPCR检测正常未转染细胞分泌的外泌体(Exo)和CircRNA稳定转染细胞分泌的外泌体(CircRNA@Exo、Man-CircRNA@Rif@Exo)中的CircRNA表达水平,结果显示,CircRNA稳定转染细胞的外泌体中的CircRNA表达水平显著高于正常对照组(图6),由此可以确认外泌体能成功装载CircRNA。
通过Western blot检测单纯负载CircRNA外泌体(CircRNA@Exo)、经表面靶向修饰的CircRNA和抗结核药物共负载外泌体(Man-CircRNA@Rif@Exo)中阳性蛋白和阴性蛋白的表达情况(图7),结果显示前述两种外泌体的阳性蛋白均能表达,而不表达阴性蛋白,进而能够确认前述两种外泌体的纯度。通过粒径分析仪检测前述两种外泌体的粒径大小(图8),结果显示,与单纯负载CircRNA外泌体的粒径(99.0±4.1nm)相比,经表面靶向修饰的CircRNA和抗结核药物共负载外泌体的粒径较大(114.4±8.3nm)。通过透射电镜观察前述两种外泌体的形态(图9),结果显示两种外泌体均具有膜囊泡结构。因此,以上三种方法联合确认了经表面靶向修饰的CircRNA和抗结核药物共负载外泌体被成功制备。
实施例3
通过本实施例对实施例2所制得细胞外囊泡载药***(经表面靶向修饰的CircRNA和抗结核药物共负载外泌体)增强细胞靶向性进行验证,具体操作如下:
利用亲脂性染料DIO对实施例2成功制备的经表面靶向修饰的CircRNA和抗结核药物共负载外泌体(以不进行修饰的CircRNA和药物共负载外泌体作为对照)进行染色示踪,取1mL的实施例2制得经表面靶向修饰的CircRNA和抗结核药物共负载外泌体(1mg/mL),加入10μL的DIO(1mM),颠倒混匀,置于37℃避光孵育30min,利用100KD超滤管将游离的染料洗去,每次14000×g离心10min,加入PBS洗涤,重复离心6次。通过紫外分光光度法检测最后一次洗涤的流穿液中是否含有DIO染料(吸光度为480nm),直至确认染料洗涤完全。取500μL的PBS重悬超滤管中收集到的染色外泌体,将收集的染色外泌体均匀地分成两管,即每管体积为250μL。其中一管加入250μL的DSPE-PEG-MAN(20μg/mL),另一管加入250μL的DSPE-mPEG(20μg/mL)作为对照组,颠倒混匀,置于4℃避光孵育过夜。利用100KD超滤管将游离的DSPE-PEG-MAN或DSPE-mPEG洗去,每次14000×g离心10min,加入PBS洗涤,重复离心3次。最后取200μL的PBS分别重悬超滤管中收集到的经DSPE-PEG-MAN表面修饰后的染色外泌体和经DSPE-mPEG表面修饰后的染色外泌体,置于4℃避光保存。
(3)计数4×105个THP-1巨噬细胞铺板,在37℃、含5%CO2的环境下培养24h,分别取50μL步骤(2)得到的经DSPE-PEG-MAN表面修饰后的染色外泌体和经DSPE-mPEG表面修饰后的染色外泌体(200μg/mL),与THP-1巨噬细胞在37℃培养箱分别共孵育1h、3h和6h后,用PBS洗涤细胞2次,用胰酶消化、收集细胞,1000rpm离心5min,弃去上清,加入1mL的PBS重悬、洗涤沉淀,1000rpm离心5min,重复2次,最后收集沉淀到流式管中,加入500μL的PBS重悬细胞,利用流式细胞仪检测THP-1巨噬细胞内的荧光值MFI。
在本实施例中,以正常未转染细胞(NC-Exo)为正常对照组,利用流式细胞仪检测THP-1巨噬细胞内的荧光值MFI,其结果显示,本实施例制得经表面靶向修饰的CircRNA和抗结核药物共负载外泌体(Man-Exo)能被巨噬细胞摄取得更多,有利于该经表面靶向修饰的CircRNA和抗结核药物共负载外泌体在宿主细胞发挥调控作用(图10-11)。
实施例4
本实施例提供了一种细胞外囊泡载药***(CircRNA和抗结核药物共负载微囊泡)的制备方法包括如下步骤:
(1)通过设计和构建特异性CircRNA(以具有抗结核免疫调控功能的CircRNA—circTRAPPC6B为例)的高表达慢病毒,利用慢病毒转染293T细胞。取24孔板每孔计数1×105个细胞铺板,培养24h后以换液的方式加入500μL含2×106TU的慢病毒(MOI=10)的含血清培养基(取10μL慢病毒(1×108TU/mL)加入到490μL的含血清培养基中),继续培养48h。转染48h后在荧光显微镜下可见明显的绿色荧光表达,继续培养3-4天,用胰酶消化、收集细胞,1000rpm离心5min,弃上清,用1mL的PBS重悬沉淀,重复离心2次,最后用500μL的PBS重悬细胞沉淀,收集到流式管中。通过流式分选仪器,分选出所有含绿色荧光的细胞,分选效率为89%,从而筛选出所有含绿色荧光的CircRNA稳定高表达的细胞系;
(2)在37℃、含5%CO2的环境下分别培养正常未转染(正常对照组)、CircRNA稳定高表达的两种细胞系,取10cm培养皿每皿分别计数4×106个细胞数铺皿,培养24h后,以换液的方式加入5mL含高浓度利福平(终浓度为1mg/mL)的无血清培养基(取50μL的浓度为100mg/mL的利福平加入到4950μL的无血清培养基中),将细胞与利福平共孵育24h以诱导细胞凋亡,收集细胞培养上清。通过4℃600×g离心10min,收集离心后上清;4℃2700×g离心10min,收集离心后上清;4℃14000×g离心2min,收集离心后上清;4℃14000×g离心60min,弃去上清,用PBS洗涤沉淀3次,每次4℃14000×g离心60min,最后一次用500μL的PBS重悬沉淀,即获得CircRNA和抗结核药物共负载微囊泡,并于-80℃保存。
本实施例中,通过粒径分析确认正常未转染细胞产生的细胞微囊泡(MPs)和CircRNA稳定转染细胞产生的细胞微囊泡(CircRNA@MPs)的粒径大小(图12),其结果确认通过本实施例上述制备步骤能成功提取两种微囊泡(正常未转染细胞产生的微囊泡和CircRNA稳定转染细胞产生的微囊泡)。通过RT-qPCR检测两种微囊泡中CircRNA的表达水平,结果显示,CircRNA稳定转染细胞的微囊泡中的CircRNA表达水平显著高于正常对照组(图13),由此能够确认细胞微囊泡能成功装载CircRNA。再通过紫外分光光度法(480nm)检测细胞微囊泡的药物浓度(图14),由此能够确认细胞微囊泡能成功装载抗结核药物(利福平)。
综上,本发明以细胞外囊泡作为CircRNA和抗结核药物的共载体,高效装载抗结核免疫调控功能CircRNA和抗结核药物,再通过对细胞外囊泡进行表面靶向修饰能够进一步增强该细胞外囊泡载药***的靶向性,进而得到所述细胞外囊泡载药***。该细胞外囊泡载药***通过结合CircRNA的抗结核免疫调控功能和抗结核药物的结核分歧杆菌抑制作用,更加有效杀伤和清除宿主细胞内的结核分歧杆菌,从而有望为安全高效的抗结核药物开发提供新策略。
上述的具体实施例是对本发明技术方案和有益效果的进一步说明,并非对实施方式的限定。对本领域技术人员来说,在不脱离本发明构思的前提下任何显而易见的替换均在本发明的保护范围之内。
Claims (10)
1.一种细胞外囊泡载药***,其特征在于:包括细胞外囊泡以及负载于细胞外囊泡内的CircRNA和抗结核药物。
2.根据权利要求1所述的一种细胞外囊泡载药***,其特征在于:所述抗结核药物为利福平。
3.根据权利要求1所述的一种细胞外囊泡载药***,其特征在于:所述细胞外囊泡为外泌体或微囊泡。
4.根据权利要求1所述的一种细胞外囊泡载药***,其特征在于:所述CircRNA为circTRAPPC6B。
5.根据权利要求1-3中任意一项所述的一种细胞外囊泡载药***的制备方法,其特征在于:包括如下步骤:
(1)提取高表达CircRNA外泌体:
(2)采用单纯孵育法或电穿孔法将抗结核药物装载到步骤(1)制备的高表达CircRNA外泌体中;
(3)洗去游离的抗结核药物,加入PBS洗涤,多次离心直至完全去除游离的抗结核药物,再用PBS重悬超滤管收集得到CircRNA和抗结核药物共负载外泌体;
(4)对步骤(3)获得CircRNA和抗结核药物共负载外泌体进行表面靶向修饰,即得到所述细胞外囊泡载药***。
6.根据权利要求5所述的一种细胞外囊泡载药***的制备方法,其特征在于:在步骤(2)中,所述抗结核药物为利福平,所述电穿孔法的具体步骤为:取100μL的步骤(1)制备浓度为1mg/mL的高表达CircRNA外泌体于EP管中,加入100μL的浓度为2mg/mL的利福平,颠倒混匀置于冰上,设置电穿孔仪器的操作条件为1000V、10ms和2pulses,将200μL的高表达CircRNA外泌体和利福平混合物进行电穿孔,并收集电穿孔后的外泌体,置于37℃孵育30min以恢复外泌体膜结构。
7.根据权利要求5所述的一种细胞外囊泡载药***的制备方法,其特征在于:在步骤(4)中,表面靶向修饰的具体操作为:取1mL的步骤(3)制备的浓度为500μg/mL的CircRNA和抗结核药物共负载外泌体,加入1mL的浓度为20μg/mL的DSPE-PEG-MAN,颠倒混匀,置于4℃摇床避光孵育过夜;再利用超滤管将游离的DSPE-PEG-MAN洗去,加入PBS洗涤,重复离心多次。
8.根据权利要求1-3中任意一项所述的一种细胞外囊泡载药***的制备方法,其特征在于:包括如下步骤:
(S1)培养并筛选含CircRNA稳定高表达的细胞系;
(S2)将步骤(S1)所得的细胞系与抗结核药物共孵育以诱导细胞凋亡,收集细胞培养上清液,重复离心、沉淀多次后,再用PBS重悬沉淀,即得到所述细胞外囊泡载药***。
9.如权利要求1-4任意一项所述的细胞外囊泡载药***在抗结核药物的应用。
10.如权利要求6-7任意一项所述的细胞外囊泡载药***的制备方法在制备抗结核药物的应用。
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