CN111973761A - 一种具有肿瘤诊疗功能的外泌体及其制备方法与应用 - Google Patents
一种具有肿瘤诊疗功能的外泌体及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供一种具有肿瘤诊疗功能的外泌体及其制备方法与应用,属于纳米生物医学技术领域。为了解决肿瘤治疗时药物载体靶向性弱,药物载体生物相容性差的问题,本发明利用双配体靶向修饰,将细胞穿透肽TAT修饰的稀土掺杂碳点装载到细胞靶向肽RGD修饰的外泌体中所得到具有诊疗肿瘤功能的外泌体,其能在肿瘤部位富集并对肿瘤细胞进行光热消融,同时还可以实现肿瘤磁共振成像和计算机断层扫描成像诊断。
Description
技术领域
本发明属于纳米生物医学技术领域,具体涉及一种具有肿瘤诊疗功能的外泌体及其制备方法与应用
背景技术
在肿瘤治疗领域靶向给药仍然是一个关键的问题。近年来,研究者开发了多种人工纳米颗粒用于肿瘤药物的体内递送,然而,这些外源性纳米颗粒可能在体内迅速地被免疫系统识别,易被肝脏和肾脏清除出体外。外泌体,是由活细胞产生的一类天然存在的膜囊泡,能够通过携带的蛋白质、脂质和RNA等物质参与细胞间通讯和物质交换。同时,外泌体也可通过增强的渗透和保留(EPR)效应向肿瘤内扩散,被认为是天然的药物递送载体,但是外泌体的肿瘤靶向性比较弱。
光热治疗(PTT)是一种基于光热试剂的新兴肿瘤治疗方法,光热试剂能够将近红外光能转化为热,产生局部高热杀死肿瘤细胞。外泌体与光热治疗相结合的策略为肿瘤治疗提供了新的途径。
多巴胺,天然存在于生物体内,可被碳化形成碳点(CDs),并在碳点表面自聚合形成聚多巴胺(PDA),一种广泛应用的肿瘤光热治疗试剂。碳点中掺杂稀土元素Gd和Dy,可以实现肿瘤核磁共振(MRI)和计算机断层扫描(CT)成像诊断。因此,将上述碳点封装到外泌体中可以实现同时的肿瘤光热治疗和MRI/CT成像诊断,在肿瘤临床治疗上具有重要意义,但其肿瘤靶向性需提高。
发明内容
本发明的目的是为了解决肿瘤治疗时药物载体靶向性弱和药物载体生物相容性差的问题,本发明提供了一种具有肿瘤诊疗功能的外泌体,所述的外泌体是将细胞穿透肽TAT修饰的稀土掺杂碳点装载到细胞靶向肽RGD修饰的外泌体中所得到的。
在本发明的一个实施例中,所述的外泌体是从RAW264.7细胞中提取获得的。
在本发明的一个实施例中,所述的外泌体的尺寸(直径)是79±6纳米。
在本发明的一个实施例中,所述的靶向肽RGD的氨基酸组成是:Arg-Gly-Asp。
在本发明的一个实施例中,所述的TAT的氨基酸序列是YGRKKRRQRRR,如SEQ IDNO.1所示。
本发明还提供了一种具有肿瘤诊疗功能的外泌体的制备方法,所述的制备方法是将细胞穿透肽TAT修饰的稀土掺杂碳点装载到细胞靶向肽RGD修饰的外泌体中,得到具有肿瘤诊疗功能的外泌体。
在本发明的一个实施例中,所述的外泌体的制备方法包括:
1)将RAW264.7细胞通过含有肿瘤靶向肽RGD的培养液培养,收集培养液上清,通过超速离心法得到靶向肽RGD修饰的外泌体Exo-RGD;
2)以多巴胺为碳源,加入稀土元素得到稀土掺杂碳点CDs:Gd,Dy,然后在表面进行细胞穿透肽TAT修饰,得到细胞穿透肽TAT修饰的稀土掺杂碳点CDs:Gd,Dy-TAT;
3)将步骤2)得到的CDs:Gd,Dy-TAT装载到步骤1)得到的Exo-RGD中,得到具有肿瘤诊疗功能的外泌体CDs:Gd,Dy-TAT@Exo-RGD。
进一步地限定,所述的细胞穿透肽TAT修饰的方法是:取EDC和NHS加入到CDs:Gd,Dy溶液中在室温条件下活化,在反应液中加入细胞穿透肽TAT继续反应,将反应液透析后得到细胞穿透肽TAT修饰的稀土掺杂碳点CDs:Gd,Dy-TAT。
进一步地限定,所述的装载是通过超声方式完成的,所述的超声方式的设置参数为:振幅20%,3分钟6次循环,间隔30秒,每次循环之间设置2分钟冷却时间。
本发明还提供了上述具有肿瘤诊疗功能的外泌体在制备肿瘤诊疗药物中的应用。
有益效果
本发明创造性地设计了双配体靶向修饰,是将细胞穿透肽TAT修饰的稀土掺杂碳点装载到细胞靶向肽RGD修饰的外泌体中所得到的具有诊疗肿瘤功能的外泌体,其能在肿瘤部位富集并对肿瘤细胞进行杀伤,达到有效治疗肿瘤的效果,同时上述的外泌体还具备MRI和CT成像诊断功能。
附图说明
图1为透射电镜图,其中a是CDs:Gd,Dy,b是RGD-Exo,c是CD:Gd,Dy-TAT@Exo-RGD;
图2为检测Exo、Exo-RGD和CD:Gd,Dy-TAT@Exo-RGD中标志蛋白的表达情况的免疫印迹结果图,其中1是Exo,2是Exo-RGD,3是CD:Gd,Dy-TAT@Exo-RGD;
图3为测定CD:Gd,Dy-TAT@Exo-RGD的体外光热转换效率结果图,其中a是不同样品的体外光热转换效率结果图,b是不同浓度的CD:Gd,Dy-TAT@Exo-RGD体外光热转换效率结果图;
图4为测定CD:Gd,Dy-TAT@Exo-RGD的体外光热治疗效率结果图,其中a是未经激光照射的HeLa细胞存活率,b是未经激光照射的4T1细胞存活率,c是经激光照射的HeLa细胞存活率,d是经激光照射的4T1细胞存活率;
图5为CDs:Gd,Dy-TAT@Exo-RGD在体内肿瘤治疗效果图;
图6为CDs:Gd,Dy-TAT@Exo-RGD体内成像性能结果图,a是小鼠的T1-MRI成像图,b是小鼠的T2-MRI成像图,c是CT成像图;
图7为Balb/c小鼠主要器官的组织切片图;
图8为Balb/c小鼠血液生化分析结果图,其中图a是天冬氨酸转氨酶(AST),图b是丙氨酸转氨酶(ALT),图c是尿酸(UA),图d是肌酐(CREA),图e是白细胞(WBC),图f是红细胞(RBC),图g是血红蛋白(HGB),图h是红细胞压积(HTC),图i是血小板(PLT)。
具体实施方式
多巴胺盐酸盐DA、氯化钆GdCl3·6H2O、氯化镝DyCl3·6H2O、MTT、NHS、EDC、和PBS是从SigmaAldrich公司购买。
RGD肽和TAT肽是从南京肽业生物科技有限公司购买。
RIPA裂解液、BCA试剂盒和ECL化学发光试剂盒是从碧云天生物技术有限公司购买。
抗体CD63、抗体CD81和抗体TSG101是从Abcam公司购买。
HeLa细胞系、Raw 264.7细胞系和4T1细胞系是从中国科学院上海细胞库购买。
FBS、青霉素、链霉素、RPMI 1640培养基和DMEM培养基是从Gibco公司购买。
多聚甲醛和水合氯醛是从阿拉丁试剂公司购买。
实施例1.
一、CD:Gd,Dy-TAT@Exo-RGD的制备
1)提取Exo-RGD外泌体
将RAW264.7细胞通过含有肿瘤靶向肽RGD的培养液培养,收集培养液上清,通过超速离心法得到靶向肽RGD修饰的外泌体Exo-RGD,具体方法如下:
将RAW264.7细胞用含10%FBS、1%青霉素和链霉素的RMPI 1640培养基于37℃,5%CO2环境中培养,培养至80%融合度后,加入30mL无外泌体血清FBS的RMPI 1640培养基继续培养,并加入靶向肽RGD(100μg/mL),继续培养48小时后,收集培养液上清。
将收集的上清液进行差速离心:首先在4℃条件下500g离心10分钟收集上清液,将上清液在4℃条件下2000g离心10分钟除去细胞碎片并收集上清液,将上清液在4℃条件下10000g离心30分钟,然后将上清液经0.22μm滤膜除去细胞与细胞碎片并收集上清液,在4℃条件下120000g离心1小时重悬沉淀,最后将重悬沉淀后的产物在4℃条件下120000g离心1小时,将离心后的产物重悬于200μL 1×PBS中,即得到Exo-RGD,将其分装后在-80℃冰箱内保存。
2)制备CDs:Gd,Dy-TAT
以多巴胺为碳源,加入稀土元素得到稀土掺杂碳点CDs:Gd,Dy,然后在表面进行细胞穿透肽TAT修饰,得到细胞穿透肽TAT修饰的稀土掺杂碳点CDs:Gd,Dy-TAT,具体方法如下:
制备稀土掺杂的碳点(CDs:Gd,Dy):取500mg多巴胺盐酸盐(DA),溶于10mL水中,将此多巴胺水溶液转移到聚四氟乙烯反应釜中在180℃条件下反应8小时,待反应液的温度降到室温后,将反应液于室温条件下11000rpm离心10分钟,然后将离心后的产物用2/3的去离子水和1/3的75%乙醇混合液洗涤三次,将洗涤后的产物进行去离子水透析24小时,并经0.22μm滤器过滤,将过滤后的样品冻干,得到稀土掺杂的碳点CDs:Gd,Dy。
连接细胞穿透肽TAT(CDs:Gd,Dy-TAT):取10mg EDC和10mg NHS加入到10mL CDs:Gd,Dy溶液中,在室温条件下活化1小时,然后在反应液中加入12mg细胞穿透肽TAT,继续反应12小时,将反应液透析24小时除去未反应的细胞穿透肽TAT,得到CDs:Gd,Dy-TAT。
3)外泌体负载
将步骤2)得到的CDs:Gd,Dy-TAT装载到步骤1)得到的Exo-RGD中,得到具有肿瘤诊疗功能的外泌体CDs:Gd,Dy-TAT@Exo-RGD,具体方法如下:
在10mM PBS溶液中加入100μg RGD-Exo和100μLCDs:Gd,Dy-TAT,将反应液加入到离心管中,将离心管置于超声装置中,设置参数如下:振幅20%,3分钟6次循环,间隔30秒,每次循环之间设置2分钟冷却时间,进行超声载药,超声处理后将反应液置于37℃培养箱中孵育60分钟以恢复外泌体膜结构,再将反应液在室温条件下120000g超速离心60分钟后弃上清液得到负载CD:Gd,Dy-TAT药物的外泌体沉淀即CD:Gd,Dy-TAT@Exo-RGD,最后,将沉淀重新分散到1×PBS中,在4℃条件下保存备用。
二、CD:Gd,Dy-TAT@Exo-RGD的表征
透射电镜分析:将Exo-RGD、CD:Gd,Dy-TAT和CD:Gd,Dy-TAT@Exo-RGD分别分散在水中,充分分散后,将产物滴于碳支持膜上,通过透射电镜仪器获得以上材料的形态和尺寸,结果如图1所示,以上材料都具有比较均匀的粒子尺寸,如图1中a所示,CD:Gd,Dy-TAT尺寸(直径)是9纳米,如图1中b所示,Exo-RGD的尺寸(直径)是70±9纳米,如图1中c所示,CD:Gd,Dy-TAT@Exo-RGD的尺寸(直径)79±6纳米。
蛋白免疫印迹分析:将分散在1×PBS中的Exo、Exo-RGD、CDs:Gd,Dy-TAT@Exo-RGD与RIPA裂解液混合,经过充分裂解后,在4℃条件下12000g离心10分钟后吸上清液即为蛋白溶液,用BCA试剂盒测定蛋白浓度,用10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离蛋白质样品,分离后转移到固相载体聚偏氟乙烯(PVDF)膜上,在室温下,将PVDF膜用5%的脱脂奶粉封闭1小时,以防止非特异性结合,然后将PVDF膜与CD63、CD81、TSG101一抗4℃孵育过夜,次日,用TBST洗涤液漂洗膜3次,每次10分钟。随后,将PVDF膜与二抗在室温下孵育1小时,最后,使用ECL化学发光试剂盒对膜上的蛋白质进行可视化和拍照。结果如图2所示,Exo、Exo-RGD、CDs:Gd,Dy-TAT@Exo-RGD均显示特征膜蛋白CD63,CD81和TSG101的表达,说明在RGD改性和超声封装药物之后,外泌体的结构和功能没有改变。
三、测定CDs:Gd,Dy-TAT@Exo-RGD体外光热转换效率
分别将1mL浓度为200μg mL-1的CDs:Gd,Dy-TAT、Exo-RGD和CDs:Gd,Dy-TAT@Exo-RGD水溶液放置在石英皿中,纯水作为对照组,用808nm的激光器照射,溶液的温度变化用数字温度计测定,每30秒测定一次,在激光照射过程中,使用红外相机来检测温度变化,为了测定样品浓度对光热效率的影响,采用不同浓度(0、50、100、200和400μg mL-1)的CDs:Gd,Dy-TAT@Exo-RGD样本水溶液经808nm的激光照射,同时监测温度变化。同样地,也测定了激光能量密度对光热效率的影响,结果如图3中a所示,在激光照射下,CDs:Gd,Dy,CDs:Gd,Dy-TAT和CDs:Gd,Dy-TAT@Exo-RGD水溶液温度随照射时间延长迅速增高,从环境温度25℃分别升高至63.6℃,63.5℃和63.8℃左右,而纯水和Exo-RGD在相同的激光照射条件下却无明显升温,证实了CDs:Gd,Dy-TAT@Exo-RGD具有优越的光热转换特性;如图3中b所示,随CDs:Gd,Dy-TAT@Exo-RGD浓度升高,细胞存活率显著下降,正常条件下,癌细胞在42℃便会发生凋亡,所以上述结果证实CDs:Gd,Dy-TAT@Exo-RGD溶液在激光照射下温度能够超过60℃,说明其具有良好的光热治疗潜力。
四、测定CDs:Gd,Dy-TAT@Exo-RGD在体外的光热治疗效率
通过MTT法测定材料对细胞的毒性,选用4T1和HeLa两种细胞系,4T1细胞用含有10%FBS、1%青霉素和链霉素的RPMI 1640培养基于37℃,5%CO2环境中培养,HeLa细胞用含有10%FBS、1%青霉素和链霉素的DMEM培养基于37℃,5%CO2环境中培养,首先,采用不同浓度(0、100、200、400和800mgmL-1)的CDs:Gd,Dy,CDs:Gd,Dy-TAT,Exo-RGD和CDs:Gd,Dy-TAT@Exo-RGD分别和两种细胞共孵育24小时后,弃掉培养基,用1×PBS洗涤两次,随后,加入新鲜培养基,并在808nm激光器下照射5分钟,继续37℃培养24小时,最后通过MTT法测定细胞存活率,未经过激光照射的细胞选作对照组,结果如图4中a和图4中b所示,未经激光照射,在CDs:Gd,Dy,CDs:Gd,Dy-TAT,Exo-RGD和CDs:Gd,Dy-TAT@Exo-RGD浓度为800μg mL-1时,共孵育的HeLa细胞和4T1细胞存活率都高于90%,说明在没有激光照射时材料对细胞毒性非常低;结果如图4中c和图4中d所示,经激光照射之后,无论是HeLa细胞还是4T1细胞的存活率都随CDs:Gd,Dy,CDs:Gd,Dy-TAT,Exo-RGD和CDs:Gd,Dy-TAT@Exo-RGD的浓度增加而下降,具体的,在浓度为800μg mL-1时,与CDs:Gd,Dy-TAT@Exo-RGD共孵育的细胞存活率都低于10%,显著高于CDs:Gd,Dy和CDs:Gd,Dy-TAT的30%和16%,不仅说明了CDs:Gd,Dy-TAT@Exo-RGD具有优异的光热治疗效果,同时还证实了双配体肿瘤靶向性。
五、验证CDs:Gd,Dy-TAT@Exo-RGD体内肿瘤治疗效果
选取8周龄Balb/c雌性小鼠建造小鼠肿瘤模型,5×106个4T1细胞悬于100μL 1×PBS中,皮下注射于小鼠右后肢上部,待肿瘤长到60立方毫米时,肿瘤鼠随机分成五个组分:(1)静脉注射100μL1×PBS(2)NIR激光照射10分钟(3)静脉注射100微升CDs:Gd,Dy,NIR激光照射10分钟(4)静脉注射100微升CDs:Gd,Dy-TAT,用NIR激光照射10分钟(5)静脉注射100μLCDs:Gd,Dy-TAT@Exo-RGD,用NIR激光照射10分钟,每隔一天检测一次肿瘤体积和小鼠体重,结果如图5所示,对于PBS和NIR组,随着时间的增加,小鼠肿瘤体积明显增大,说明单独的NIR激光照射不能够抑制肿瘤生长,CDs:Gd,Dy+NIR组小鼠肿瘤的生长速率相比前两组明显降低,CDs:Gd,Dy-TAT+NIR组小鼠肿瘤生长在一定程度上被抑制,而CDs:Gd,Dy-TAT@Exo-RGD+NIR组小鼠肿瘤生长被明显抑制,处理后3天,肿瘤开始缩小,第21天即被完全消融,在小鼠体内证实了CDs:Gd,Dy-TAT@Exo-RGD具有良好的肿瘤靶向性和光热治疗效果。
六、验证CDs:Gd,Dy-TAT@Exo-RGD在体内成像性能
MRI成像:肿瘤小鼠静脉注射CDs:Gd,Dy,CDs:Gd,Dy-TAT和CDs:Gd,Dy-TAT@Exo-RGD溶液100μL,在成像之前腹腔注射100μL 10%水合氯醛溶液将小鼠麻醉,通过GEDiscovery MRI 7503.0T成像系统进行MRI成像,T1-MRI参数如下:FOVread=210×210mm,TE=21.0ms,TR=570ms,切片厚度:1.9mm,T2-MRI参数为:FOVread=230×230mm,FOVread=200×200,TE=110ms,TR=4000ms,切片厚度:1.8mm。
CT成像:肿瘤小鼠静脉注射CDs:Gd,Dy,CDs:Gd,Dy-TAT和CDs:Gd,Dy-TAT@Exo-RGD溶液100μL,在成像之前腹腔注射100μL 10%水合氯醛溶液进行麻醉,使用PhilipsBrilliance iCT 256型仪器进行CT成像,参数如下:120KVp,SW:1.0mm,FOV:72mm,Z:2.00。
结果如图6中a所示,随着时间的延长,CDs:Gd,Dy、CDs:Gd,Dy-TAT和CDs:Gd,Dy-TAT@Exo-RGD三组小鼠的肿瘤部位的T1-MRI图像明显变亮,如图6中b所示,T2-MR图像明显变暗,如图6中c所示,CT成像对比也逐渐增强,且CDs:Gd,Dy-TAT@Exo-RGD的对比效果最强。
七、验证CDs:Gd,Dy-TAT@Exo-RGD的生物安全性
组织切片分析:将Balb/c小鼠静脉注射100μLCDs:Gd,Dy、CDs:Gd,Dy-TAT、Exo-RGD和CDs:Gd,Dy-TAT@Exo-RGD溶液,经过14天后,将小鼠安乐死,取主要的器官(心,肝,脾,肺,肾)肌肉和脑,将样本固定在4%多聚甲醛溶液中48小时,再嵌入石蜡中,切成3μm片,将切片经H&E染色,最后用光学显微镜观察拍照,同时,采集不同组分的血样进行血液生化分析。结果如图7所示,各组均无明显炎症反应,细胞形态和结构正常,未见坏死,肾脏样品中的肾小球结构正常,心样品中心肌组织正常,说明制备的CDs:Gd,Dy-TAT@Exo-RGD没有明显的作用,显示出很好的生物相容性。
结果如图8所示,血液生化分析得到天冬氨酸转氨酶(AST),丙氨酸氨基转移酶(ALT),尿酸(UA)和肌酐(CREA)数据。AST和ALT水平能够反映肝功,AST和ALT升高说明存在肝损伤,不同组分的AST和ALT都在正常范围值内,证明材料不存在肝毒性。UA和CREA能够反映肾功,不同组分的UA和CREA和正常小鼠的类似,证实了本发明合成的材料不能引起肝毒性和肾毒性,同时也测定红细胞(RBC)、白细胞(WBC)、血小板(PLT)、血红蛋白(HGB)、红细胞压积(HCT)等全血指标,结果显示未发现任何异常,说明材料具有优异的生物相容性。
以上详细描述了本发明的优选方式,但是,本发明并不限于此。在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,包括各个技术特征以任何其他的合适方式进行组合,这些简单变型和组合同样当视为本发明所公开的内容,均属于本发明的保护范围。
SEQUENCE LISTING
<110> 中国农业科学院特产研究所
<120> 一种具有肿瘤诊疗功能的外泌体及其制备方法与应用
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 11
<212> PRT
<213> 细胞穿透肽TAT
<400> 1
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10
Claims (10)
1.一种具有肿瘤诊疗功能的外泌体,其特征在于,所述的外泌体是将细胞穿透肽TAT修饰的稀土掺杂碳点装载到细胞靶向肽RGD修饰的外泌体中所得到的。
2.根据权利要求1所述的外泌体,其特征在于,所述的外泌体是从RAW264.7细胞中提取获得的。
3.根据权利要求1所述的外泌体,其特征在于,所述的外泌体的尺寸是79±6纳米。
4.根据权利要求1所述的外泌体,其特征在于,所述的RGD的氨基酸组成是:Arg-Gly-Asp。
5.根据权利要求1所述的外泌体,其特征在于,所述的TAT的氨基酸序列YGRKKRRQRRR。
6.权利要求1-5任意一项所述的外泌体的制备方法,其特征在于,所述的制备方法是将细胞穿透肽TAT修饰的稀土掺杂碳点装载到细胞靶向肽RGD修饰的外泌体中,得到具有肿瘤诊疗功能的外泌体。
7.根据权利要求6所述的制备方法,其特征在于,所述的制备方法包括:
1)将RAW264.7细胞通过含有肿瘤靶向肽RGD的培养液培养,收集培养液上清,通过超速离心法得到靶向肽RGD修饰的外泌体Exo-RGD;
2)以多巴胺为碳源,加入稀土元素得到稀土掺杂碳点CDs:Gd,Dy,然后在表面进行细胞穿透肽TAT修饰,得到细胞穿透肽TAT修饰的稀土掺杂碳点CDs:Gd,Dy-TAT;
3)将步骤2)得到的CDs:Gd,Dy-TAT装载到步骤1)得到的Exo-RGD中,得到具有肿瘤诊疗功能的外泌体CDs:Gd,Dy-TAT@Exo-RGD。
8.根据权利要求7所述的制备方法,其特征在于,步骤2)中,所述的细胞穿透肽TAT修饰的方法是:取EDC和NHS加入到CDs:Gd,Dy溶液中在室温条件下活化,在反应液中加入细胞穿透肽TAT继续反应,将反应液透析后得到细胞穿透肽TAT修饰的稀土掺杂碳点CDs:Gd,Dy-TAT。
9.根据权利要求7所述的制备方法,其特征在于,步骤3)中,所述的装载是通过超声方式完成的,所述的超声方式的设置参数为:振幅20%,3分钟6次循环,间隔30秒,每次循环之间设置2分钟冷却时间。
10.权利要求1-5任意一项所述的外泌体在制备肿瘤诊疗药物中的应用。
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