CN117085142A - 一种负载小分子药物的间充质干细胞外泌体及其制备方法与应用 - Google Patents
一种负载小分子药物的间充质干细胞外泌体及其制备方法与应用 Download PDFInfo
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Abstract
本公开提供了一种负载小分子药物的间充质干细胞外泌体及其制备方法与应用,属于生物技术领域。本公开以负载小分子药物的间充质干细胞外泌体用于治疗银屑病,一方面减少自身抗原的产生以纠正免疫失衡,缓解或抑制银屑病病变部位炎症因子表达,达到治疗或缓解银屑病的作用;同时该技术可改善代谢紊乱,预防并发症的发生。本公开负载小分子药物的间充质干细胞外泌体的制备方法简单,载药率高,疗效显著。
Description
技术领域
本公开涉及细胞生物学、分子生物学及药物研发技术领域,具体涉及一种负载小分子药物的间充质干细胞外泌体及其制备方法与应用。
背景技术
银屑病是一种以T细胞介导炎症和角质形成细胞异常分化为主要特征的慢性炎症性疾病,以局限或泛发的鳞屑性红色斑块为主要临床表现,具有治疗难度大、容易复发的特点,可给患者造成较大的经济和心理负担。近年来该病的患病率及发病率逐年升高。本病发病机制复杂,目前认为与遗传、应激、精神紧张及免疫失调有关。目前医学上对银屑病的治疗手段包括系统使用阿维A、甲氨蝶呤等药物,局部外用糖皮质激素、维生素D3衍生物等。然而这些传统药物在取得疗效的同时,不可避免对其他器官造成损害如肝肾损害,且传统药物半衰期较短,易于被机体代谢,给药频繁,患者依从性较差等。因此,开发银屑病治疗药物成为当前的研究热点。
发明内容
本公开的目的在于克服现有技术的不足,提供一种负载小分子药物的间充质干细胞外泌体及其制备方法与应用。
为实现上述目的,本公开采取的技术方案为:
提供一种负载小分子药物的间充质干细胞外泌体,包括间充质干细胞外泌体以及附包裹在间充质干细胞外泌体内的小分子药物。
本公开以负载小分子药物的间充质干细胞外泌体用于治疗银屑病,一方面减少自身抗原的产生以纠正免疫失衡,缓解或抑制银屑病病变部位炎症因子表达,达到治疗或缓解银屑病的作用;同时该技术可改善代谢紊乱,预防并发症的发生。本公开负载小分子药物的间充质干细胞外泌体的制备方法简单,载药率高,疗效显著。
在一个实施方式中,所述小分子药物为缬氨酸、N-ω-羟基-L-去甲精氨酸二乙酸酯盐中的至少一种。
在一个实施方式中,所述间充质干细胞外泌体和小分子药物的质量比为(10:1)-(1:10)。
在一个实施方式中,所述间充质干细胞外泌体为人体间充质干细胞来源的外泌体。
在本说明书中,术语“间充质干细胞”是指具有能够分化为脂肪、软骨、骨、肌肉、皮肤、神经等细胞的多能性的干细胞。上述间充质干细胞可从诱导多能干细胞分化或从骨髓、脂肪组织、脐带组织、脐带血、骨骼肌、外周血、润滑膜、羊水等分离。
所述间充质干细胞为脂肪间充质干细胞,脐带来源间充质干细胞,羊膜间充质干细胞,皮肤间充质干细胞,牙髓间充质干细胞中的至少一种。
在一个实施方式中,所述间充质干细胞外泌体是通过以下方法获得:
步骤S1:在细胞培养基中培养间充质干细胞;
以及步骤S2:从间充质干细胞的培养液中分离外泌体。
在本公开的一实例中,上述步骤S2可通过实施离心分离来从间充质干细胞的培养液中分离外泌体。
在本公开的一实例中,上述步骤S2可通过实施离心分离来从间充质干细胞的培养液中分离外泌体。
更具体地,以200~400xg的条件对上述间充质干细胞的培养基进行5分钟至20分钟的离心分离,来去除剩余的细胞和细胞残留物,之后,取出上清液并以9000~12000xg的条件进行60分钟~80分钟的高速离心分离,之后,再次取出上清液并以90000~120000xg的条件进行80分钟~100分钟的超高速离心分离,由此去除上清液,从而可获取留在底层的外泌体。
根据本公开的特定实例,回收间充质干细胞培养基,在4℃、300xg的条件下进行10分钟的离心分离来去除剩余的细胞;之后,取出上清液并在4℃、2000xg的条件下进行20分钟~80分钟的离心来去除死亡细胞;之后,取出上清液并在4℃、10000xg的条件下进行20分钟~80分钟的离心来去除细胞碎片;取出上清液并利用0.22μm的过滤器进行过滤;之后,利用超速离心机在120000xg、4℃的条件下进行90分钟的离心分离;之后,去除上清液,所得沉淀加入10mL PBS重悬;所得重悬液在4℃,120000xg条件下离心90min,弃上清,保留沉淀,沉淀即为间充质干细胞来源的外泌体,用100μL PBS重悬,-80℃冰箱保存待用。
在本公开的一个实施方式中,所述细胞培养基选自BasalMedia的MEMα培养基。
一种负载小分子药物的间充质干细胞外泌体的制备方法,通过孵育的方法将小分子药物包裹入间充质干细胞外泌体。
在本公开的一个实施方式中,所述孵育的方法主要按以下步骤进行:
步骤S1:将一定浓度的外泌体悬浮液和药物溶液混合,置于一定温度下孵育一定时间;
步骤S2:随后将上述溶液置于一定截留分子量的超滤管中,利用生物相容性介质超滤洗涤三次,得到载药的外泌体。
本方法中,外泌体悬液的浓度范围为1×106/mL–1×1012/mL,优选为1×1010/mL;孵育温度的范围为4℃–50℃,优选为37℃;孵育时间范围为1h–48h,优选为4h;超滤管的截留分子量范围为10KDa–5000KDa,优选为100KDa;生物相容性介质为生理盐水,生理缓冲液,细胞培养基等。
在本公开的一个实施方式中,通过常规的超滤、超速离心或者脱盐柱等手段将包裹药物小分子药物的外泌体与游离的药物小分子药物分离。
负载小分子药物的间充质干细胞外泌体在制备银屑病用药物中的应用。
一种用于治疗银屑病的药剂学组合物,所述药剂学组合物包含利用负载小分子药物的间充质干细胞外泌体作为有效成分。
在本说明书中使用的术语“治疗”是指(a)抑制银屑病的发展;(b)减轻银屑病;以及(c)去除银屑病。
根据本公开的优选实例,本公开的组合物为(a)如上所述的本公开的利用干扰素γ预处理的诱导多能干细胞来源的间充质干细胞或其培养物的药剂学有效量;以及(b)包含药剂学上可接受的载体的药剂学组合物。在本说明书中,术语“药剂学有效量”是指达成如上所述的诱导多能干细胞来源的间充质干细胞或其培养物的功效或活性的充分量。
在本公开的组合物制备为药剂学组合物的情况下,本公开的药剂学组合物包含药剂学上可接受的载体。本公开的药剂学组合物所包含的药剂学上可接受的载体包括通常用于制剂的乳糖、右旋糖、蔗糖、山梨糖醇、甘露醇、淀粉、阿拉伯树胶、磷酸钙、海藻酸盐、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石粉、硬脂酸镁及矿物油等,但并不限定于此。除上述成分之外,本公开的药剂学组合物还可包括润滑剂、保湿剂、甜味剂、调味剂、乳化剂、悬浮剂、防腐剂等。在雷明登氏药学全书(Remington's Pharmaceutical Sciences)(19th ed.,1995)详细记载了适合的药剂学上可接受的载体及制剂。
本公开的药剂学组合物可口服给药或肠胃外给药,例如,可通过静脉注射、皮下注射、肌肉注射、腹腔注射、局部给药、鼻腔给药、肺内给药、直肠内给药、鞘内给药、眼部给药、皮肤给药及经皮给药等的方法给药。
本公开的药剂学组合物的适合剂量可根据制剂化方法、给药方式、患者年龄、体重、性别、病态、饮食、给药时间、给药路径、排泄速度及反应灵敏度的因素处方的不同。本公开的药剂学组合物的常规剂量以成人为基准在0.0001~1000mg/kg的范围以内,但并不限定于此。
在本公开的一实例中,本公开的药剂学组合物的剂量可以为0.001~1000mg/kg、0.01~1000mg/kg、0.1~1000mg/kg、1~1000mg/kg、5~1000mg/kg、10~1000mg/kg、20~1000mg/kg、30~1000mg/kg、50~1000mg/kg、100~1000mg/kg、0.0001~100mg/kg、0.001~100mg/kg、0.01~100mg/kg、0.1~100mg/kg、1~100mg/kg、5~100mg/kg、10~100mg/kg、20~100mg/kg、30~100mg/kg或50~100mg/kg,更具体地,可以为1mg/kg、5mg/kg、10mg/kg、15mg/kg、20mg/kg、25mg/kg、30mg/kg、35mg/kg、40mg/kg、45mg/kg、50mg/kg、55mg/kg、60mg/kg、65mg/kg、70mg/kg、75mg/kg、80mg/kg、85mg/kg、90mg/kg、95mg/kg、100mg/kg、150mg/kg、200mg/kg、250mg/kg、300mg/kg、350mg/kg、400mg/kg、450mg/kg、500mg/kg、750mg/kg或1000mg/kg。
本公开的药剂学组合物可根据本公开所属技术领域的普通技术人员容易实施的方法利用药剂学上可接受的载体和/或赋形剂来制剂化,从而以单位容量形态制备或向多容量容器内添加来制备。在此情况下,剂型可以为油介质或水性介质中的溶液、悬浮液、糖浆剂或乳化液形态或提取剂、散剂、粉末剂、颗粒剂、片剂或胶囊剂形态,还可包括分散剂或稳定化剂。
与现有技术相比,本公开的有益效果为:本公开以负载小分子药物的间充质干细胞外泌体作为治疗银屑病,可以减少自身抗原的产生,可以缓解或抑制银屑病病变部位炎症因子表达,达到治疗或缓解银屑病的作用;同时改善代谢紊乱,预防并发症的发生;本公开负载小分子药物的间充质干细胞外泌体的制备方法简单,载药率高。
附图说明
图1为脐带间充质干细胞表面标记分子的测定图;
图2为脐带间充质干细胞成脂分化检测图,其中,图2A为加入成脂诱导液的实验组,图2B为加入常规培养基的对照组;
图3为脐带间充质干细胞成骨分化检测图,其中,图3A为加入成脂诱导液的实验组,图3B为加入常规培养基的对照组;
图4为脐带间充质干细胞外泌体的透射电镜图和粒径分布图,其中,图4A为脐带间充质干细胞外泌体的透射电镜图,图4B为脐带间充质干细胞外泌体的粒径分布图;
图5为本公开银屑病模型的建立方法;
图6为本公开不同处理下小鼠背部皮损变化和病理切片;
图7为本公开银屑病小鼠模型各组皮肤厚度程度(thickness intensity)评分、磷屑程度(scalling intensity)评分、体重变化程度评分、红斑程度(erythema)评分;
图8为本公开银屑病小鼠模型各组脾脏组织和脾指数。
具体实施方式
为了更好地说明本公开的目的、技术方案和优点,下面将结合具体实施例及对比例对本公开作进一步说明,其目的在于详细地理解本公开的内容,而不是对本公开的限制。本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本公开的保护范围。本公开实施所涉及的实验试剂及仪器,除非特别说明,均为常用的普通试剂及仪器。
本公开的细胞培养基采购自BasalMedia的MEMα培养基。
实施例1
1、脐带间充质干细胞的分离和培养
(1)脐带间充质干细胞(hUC-MSCs)的分离
取正常足月产健康新生儿脐带(脐带来源于健康状态良好、妊娠足月产妇自愿捐献),在GMP实验室生物安全柜中消毒后,剪成1mm×1mm×1mm大小长小段,去除脐带表皮、动脉及静脉,剥离华通氏胶并剪成细碎块状,洗涤离心后,加适量无血清培养基混匀成组织匀浆,将1mL组织匀浆接种到T75培养瓶中,加9mL/瓶培养基,放入37℃、5%二氧化碳培养箱中培养。倒置显微镜每天观察细胞生长情况。
组织贴壁培养至第5~6d,进行第一次换液,之后使用倒置显微镜每天观察细胞生长情况,根据细胞生长情况换液。一般组织匀浆接种7~10d后,可见细胞贴壁,圆形或短梭形,大小不一,之后细胞逐渐伸展呈长梭形,漩涡状生长,第14-17d细胞融合度达到70%~80%时,使用胰酶消化收获P0代细胞,洗涤离心后,按5000-6000个/cm2接种密度传P1代扩增培养,细胞培养时间为72h±12h,当P1代细胞融合度达到85%时,消化传P2代,P2代细胞融合度达到85%时,消化收获冻存为种子细胞。冻存前按照P2代种子细胞质量检测标准进行检测,结果符合要求后将部分P2代种子细胞入库,部分P2代种子细胞同前操作进行传代扩增培养,培养至P4代细胞后,将P4代细胞冻存并作为种子用于后续外泌体的提取。
(2)脐带间充质干细胞扩增培养
夹取5~10片微载片投入旋转培养瓶中,加入10mL无血清完全培养基,温和搅拌分散微载片。
从工作细胞库中取出脐带间充质干细胞,复苏后通过细胞计数取1×106个脐带间充质干细胞接种至旋转培养瓶中,加入5~10片微载片,补充培养液至最终体积为40~60mL。将混有细胞和微载体的旋转培养瓶置于37℃、5%CO2培养箱内的3D生物培养平台上,设定仪器参数为58个周期(60rpm×5min和0rpm×20min)。
培养24h后,从3D培养瓶侧壁中吸取1mL细胞悬液,检测细胞生长情况并通过细胞计数仪完成细胞计数。
72h~96h后细胞汇合度为85%左右时,更换无血清培养基,培养48h后,取细胞上清液备用,用于MSCs-Exo的提取,剩余细胞按照《剩余干细胞制剂处理措施》处理。
2、脐带间充质干细胞的鉴定
利用荧光标记的抗体CD90、CD44、CD105、CD73、CD166、CD34、CD14、CD19、CD45、HLA-DR对上述获得的脐带间充质干细胞进行染色30min,固定后,利用流式细胞仪检测脐带间充质干细胞表面标记分子表达,检测结果如图1所示。由图1可知,CD90、CD44、CD105、CD73、CD166呈阳性,CD34、CD14、CD19、CD45、HLA-DR几乎为阴性,符合脐带间充质干细胞的特征。
3、脐带间充质干细胞的成脂分化检测
在成脂培养液(含0.5mM异丁甲基黄嘌呤、60μM吲哚米辛、0.50μM氢化可的松和100μg/ml胰岛素)中诱导脐带间充质干细胞进行成脂分化。经过14天的成脂诱导,用油红O对脂滴进行染色,结果如图2所示。由图2可知,在加入成脂诱导液后,在培养7天左右时hUC-MSCs内出现脂滴,14天时通过油红O染色显示细胞内有脂性液体,hUC-MSCs向脂肪细胞分化的能力被证实。
4、脐带间充质干细胞的成骨分化检测
在成骨培养液(含1.8mM磷酸二氢钾,10nM地塞米松)中诱导脐带间充质干细胞进行成骨分化。经过28天的成骨诱导,用茜素红对钙结节进行染色,结果如图3所示。由图3可知,在加入成骨诱导液后,hUC-MSCs在7天左右向多角形转变,14天左右出现钙结节,经茜素红染色证实hUC-MSCs被诱导分化为成骨细胞,hUC-MSCs向成骨细胞分化的能力被证实。
5、脐带间充质干细胞外泌体的分离
选择脐带间充质干细胞,在培养基中培养间充质干细胞,在培养72小时后,回收间充质干细胞培养基的上清液,在4℃、300xg的条件下进行10分钟的离心分离来去除剩余的细胞;之后,取出上清液并在4℃、2000xg的条件下进行20分钟~80分钟的离心来去除死亡细胞;之后,取出上清液并在4℃、10000xg的条件下进行20分钟~80分钟的离心来去除细胞碎片;取出上清液并利用0.22μm的过滤器进行过滤;之后,利用超速离心机在120000xg、4℃的条件下进行90分钟的离心分离;之后,去除上清液,所得沉淀加入10mL PBS重悬;所得重悬液在4℃,120000xg条件下离心90min,弃上清,保留沉淀,沉淀即为间充质干细胞来源的外泌体,用100μL PBS重悬,-80℃冰箱保存待用。
6、脐带间充质干细胞外泌体的鉴定
通过投射电镜观察所分离的脐带间充质干细胞外泌体,观察结果如图4所示。采用纳米粒子分析系统检测脐带间充质干细胞外泌体的粒径,重复测量3次,通过软件进行数据分析,分析结果如图4所示,由图4可知,脐带间充质干细胞外泌体的粒径范围为50-120nm。
实施例2负载小分子药物的脐带间充质干细胞外泌体的制备
①将25μg脐带间充质干细胞外泌体与25μg的精氨酸酶抑制剂nor-NOHA混合在500μL的PBS溶液中,置于摇床中,在37℃下共孵育3小时;
②所得混合液转移至1.5mL超滤管中,在2000rpm的条件下离心5min,去除游离的小分子药物;
③所得沉淀加入500μL的PBS溶液,混匀,重复步骤②两次;
④所得沉淀加入200μL的PBS溶液,重悬沉淀,即得负载小分子药物的脐带间充质干细胞外泌体;
经茚三酮反应联合超滤测定,所得负载小分子药物的脐带间充质干细胞外泌体的载药率为29.9%。
实施例3负载小分子靶向药物小分子药物的脐带间充质干细胞外泌体的制备
①将25μg脐带间充质干细胞外泌体与25μg的缬氨酸混合在500μL的PBS溶液中,置于摇床中,在37℃下共孵育3小时;
②所得混合液转移至1.5mL超滤管中,在2000rpm的条件下离心5min,去除游离的小分子药物;
③所得沉淀加入500μL的PBS溶液,混匀,重复步骤②两次;
④所得沉淀加入200μL的PBS溶液,重悬沉淀,即得负载小分子药物的脐带间充质干细胞外泌体;
经测定,所得负载小分子药物的脐带间充质干细胞外泌体的载药率为33.2%。
实施例4应用负载小分子药物的脐带间充质干细胞外泌体治疗银屑病
银屑病小鼠模型的建立:如图1所示,选用SPF级雌性C57BL/6小鼠(6~8周龄,体质量16~18g)12只。小心剃去小鼠背部中央区域被毛,再用温和型脱毛膏脱去表面毳毛,以脊柱为中线,部脱毛区域大小约2×3cm。将小鼠随机分为2组,模型组(Imiquimod,IMQ组):每日以62.5mg的5%咪喹莫特乳膏涂抹背部皮肤;负载小分子药物的脐带间充质干细胞外泌体组(IMQ+Eng-EVs):每日以62.5mg的5%咪喹莫特乳膏涂抹背部皮肤,2小时后局部皮下多点注射含有100μg实施例2负载小分子药物的脐带间充质干细胞外泌体。实验周期连续6天,待到第7天时处死所有小鼠;观察小鼠背部皮损变化,结果如图2所示,结果显示,IMQ+Eng-EVs组与IMQ组相比,红斑消失、大多数皮肤无鳞屑覆盖、皮损与正常皮肤齐平。
银屑病小鼠模型的评估:
(1)小鼠背部皮损组织HE染色
①处死小鼠后,取背部皮肤损伤处组织,用10%甲醛溶液固定,经石蜡包埋并切片;
②将石蜡切片浸泡于二甲苯溶液中,微波炉加热5min;再次将石蜡切片浸泡入二甲苯溶液中,微波炉加热5min;再分别浸泡于无水乙醇、95%乙醇、85%乙醇、75%乙醇溶液1min、75%乙醇溶液再1min;然后用自来水进行冲洗;
③苏木素染色5min后,用流水冲洗,1%盐酸酒精分化,再次用流水冲洗;伊红染色1min;再次浸泡于75%乙醇、85%乙醇、95%乙醇、无水乙醇溶液1min、无水乙醇溶液再1min,于二甲苯溶液中浸泡5min两次后,用中性树脂封片;
④显微镜下观察并拍照,光学显微镜下观察比较各组小鼠皮肤组织病理学改变,如表皮角化程度、棘层厚度、炎性细胞浸润等,结果见图2。结果显示IMQ+Eng-EVs组可通过影响表皮细胞过度增殖、角化不全、炎症细胞浸润及血管增生而改善咪喹莫特诱导的小鼠银屑病样皮损变化。
(2)皮损评分
参考临床银屑病面积与严重性指数(PASI)评分标准,给药第1天开始,每日对背部皮损处红斑、鳞屑、厚度按0~4评分,计三项总分。PASI评分标准:(1)红斑:无红斑为0分,淡红色为1分,红色为2分,深红色为3分,极深为4分;(2)鳞屑:无鳞屑为0分,部分皮损表面上覆有鳞屑为1分,大多数皮损表面覆盖有鳞屑为2分,皮损部位几乎全部被鳞屑覆盖为3分,皮损部位全部被鳞屑覆盖为4分;(3)厚度:皮损与正常皮肤齐平为0分,皮损较正常皮肤表面稍高为1分,皮损中度隆起为2分,皮损肥厚且隆起明显为3分,皮损高度肥厚且明显凸起为4分。结果见图3。结果显示,IMQ+Eng-EVs组与IMQ组相比,皮肤厚度程度(thicknessintensity)、磷屑程度(scalling intensity)、红斑程度(erythema)显著降低,提示负载小分子药物的脐带间充质干细胞外泌体对银屑病具有很好的治疗效果。
(3)各组小鼠脾指数计算
于给药第5天,处死小鼠并取脾脏,观察脾脏组织,结果见图4。按以下公式计算脾指数:脾指数=脾质量(g)/小鼠体质量(g)。结果见图4。结果显示IMQ+Eng-EVs组小鼠脾指数降低,提示负载小分子药物的脐带间充质干细胞外泌体有效抑制了银屑病小鼠炎症过程。
最后所应当说明的是,以上实施例用以说明本公开的技术方案而非对本公开保护范围的限制,尽管参照较佳实施例对本公开作了详细说明,本领域的普通技术人员应当理解,可以对本公开的技术方案进行修改或者同等替换,而不脱离本公开技术方案的实质和范围。
Claims (9)
1.一种负载小分子药物的间充质干细胞外泌体,其特征在于,包括间充质干细胞外泌体以及附包裹在间充质干细胞外泌体内的小分子药物。
2.如权利要求1所述负载小分子药物的间充质干细胞外泌体,其特征在于,所述小分子药物为缬氨酸、N-ω-羟基-L-去甲精氨酸二乙酸酯盐中的至少一种。
3.如权利要求1所述负载小分子药物的间充质干细胞外泌体,其特征在于,所述间充质干细胞外泌体和小分子药物的质量比为(10:1)-(1:10)。
4.如权利要求1所述负载小分子药物的间充质干细胞外泌体,其特征在于,所述间充质干细胞外泌体为人体间充质干细胞来源的外泌体。
5.如权利要求1所述负载小分子药物的间充质干细胞外泌体,其特征在于,所述间充质干细胞为脂肪间充质干细胞,脐带来源间充质干细胞,羊膜间充质干细胞,皮肤间充质干细胞,牙髓间充质干细胞中的至少一种。
6.如权利要求1所述负载小分子药物的间充质干细胞外泌体,其特征在于,所述间充质干细胞外泌体是通过以下方法获得:
步骤S1:在细胞培养基中培养间充质干细胞;以及步骤S2:从间充质干细胞的培养液中分离外泌体。
7.一种如权利要求1-6任一项所述负载小分子药物的间充质干细胞外泌体的制备方法,其特征在于,通过孵育,电穿孔,挤膜,超声或冻融的方法将小分子药物包裹入间充质干细胞外泌体。
8.如权利要求1-6任一项所述负载小分子药物的间充质干细胞外泌体在制备银屑病用药物中的应用。
9.一种用于治疗银屑病的药剂学组合物,所述药剂学组合物包含利用如权利要求1-6任一项所述负载小分子药物的间充质干细胞外泌体作为有效成分。
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