CN117143231A - 包含干细胞外泌体的药物组合物及其制备方法 - Google Patents
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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Abstract
本发明涉及包含干细胞外泌体的药物组合物及其制备方法,所述外泌体通过负载化疗药物之后,能够有效的用于卵巢癌的治疗。将所述负载了化疗药物的外泌体与PI3K的单克隆抗体一起联用后,能够显著的提高化疗药物的敏感性,使得二者具有协同的显著的治疗优势,在应用前景上体现了较好的效果。
Description
技术领域
本发明涉及生物领域,具体的涉及一种包含干细胞外泌体的药物组合物及其制备方法。
背景技术
外泌体(Exosomes)是细胞分泌到胞外的一种囊泡(Extracellular Vesicles,EVs),其大小为30-150nm,具有双层膜结构,外泌体中携带有母细胞的多种蛋白质、脂类、DNA和RNA等重要信息,参与细胞间的分子传递。外泌体广泛存在于细胞培养上清以及各种体液中,包括血液、淋巴液、唾液、尿液、精液、乳汁等,同时也存在于组织样本中,如脑组织、肌肉组织、脂肪组织等。
外泌体的内容物主要取决于其细胞来源,内容繁多且成分复杂,通常包括一些生物活性物质,已知存在不同组织及细胞的外泌体内含有194种脂类,4563种蛋白质,1639种mRNAs和764种microRNAs。其中核内体分类转运复合物和四分子交联体家族成员(如CD9、CD63、cD81、cD82、cDl51)参与外泌体形成过程的分类转运。而且,有研究发现外泌体既可以表达一些MHcI类分子,HSP70、HSP90、HER2/neu和Manl,同时还可以招募多种来自于非浆膜的细胞蛋白,包括MHc分跨膜四蛋白、黏附分子和金属蛋白酶。外泌体介导细胞间的蛋白质、脂质、核酸的转运,调节受体细胞的多种生物活动,如影响基因表达。研究发现乳腺癌来源的外泌体可以调节巨噬细胞的促炎反应。在免疫系统中,一方面,树突状细胞或者肿瘤细胞的外泌体可以诱导抗肿瘤活动;另一方面,又可诱导生理状态下的免疫耐受,比如妊娠,以及在肿瘤的进展中的免疫耐受。外泌体与靶细胞膜融合后,一方面能促进供体细胞向受体细胞转运细胞表面的分子和受体;另一方面外泌体上表达的配体可以干扰细胞表面受体一配体结合,甚至可以重组受体细胞,导致受体细胞的转变。近年来研究发现外泌体在肿瘤的发生发展中发挥着重要作用,比如肿瘤的形成、进展和转移、免疫调节、免疫逃逸、血管生成、调节肿瘤转移的微环境、促进上皮-间质转化(EMT)。
外泌体也可以作为疾病治疗和药物运输的工具。基于RNA的治疗药物通过递送小干扰RNA(smallinterferingRNA,siRNA)沉默病理基因或通过将外源mRNA递送至细胞来表达治疗性蛋白质,在治疗各种疾病方面具有巨大潜力。在mRNA/siRNA输送方面,传统载体如脂质体和纳米颗粒具有无传染性,材料来源广泛,易于大量制备等优点,有着病毒载体不可替代的优势,但是它们输送效率较低、难以通过生物屏障到达病灶部位,并且易引发免疫反应被机体快速清除掉。与传统载体不同的是,一方面,外泌体包含可增强内吞作用的跨膜蛋白和膜锚定蛋白,从而促进其内部携带物质的传递;另一方面,外泌体也含有一些抑制吞噬作用的跨膜蛋白而不被循环系统快速清除,如外泌体蛋白CD47,它是一种广泛表达于细胞表面的整合素相关蛋白,可向巨噬细胞发出一种“不要吃我”的信号,保护细胞免受吞噬。学者开发了一种生物相容性肿瘤细胞-外泌体护套PSiNPs,作为靶向癌症化学疗法的药物载体。当肿瘤细胞与载有阿霉素的PSiNPs(DOX@PSiNPs)一起孵育时,肿瘤细胞便会分泌载有DOX@PSiNPs的外泌体(DOX@E-PSiNPs),DOX@E-PsiNPs对大量的癌细胞和癌症干细胞具有杀伤作用,从而起到抗肿瘤作用。外泌体也具有很多传统药物载体不具备的通过血脑屏障的能力。研究表明,脑内皮细胞产生的外泌体可作为治疗脑肿瘤药物的载体。在斑马鱼原发性脑癌模型中,装载抗癌药物的外泌体可通过血脑屏障发挥抗肿瘤作用,显著缩小肿瘤病灶大小并降低异种移植癌细胞的荧光强度和肿瘤生长标志物含量。利用基因修饰的外泌体来特异性地传递siRNA,阻止KRAS基因突变,从而使患有胰腺癌的小鼠病情得到缓解,提高小鼠的存活率,证实外泌体能够作为一种高效的RNA干扰(RNAi)载体发挥作用,运送特异性地靶向KRAS的siRNA和短发夹RNA分子,比脂质体更加高效,且没有明显的免疫反应。
卵巢癌是常见的致死性妇科恶性肿瘤之一,在女性死亡相关的癌症病因中排名第5,常常因早期诊断困难、复发率高及化疗药物的耐药性,使卵巢癌患者预后较差,5年生存率仅约30%。外泌体作为一种生物活性物质的载体,在卵巢癌的治疗中具有很大的应用前景。首先外泌体作为细胞外的一种小囊泡,可以自由循环于生物体液中,比如血液、尿液、腹水、唾液和脑脊液中。其次外泌体的膜成分中还具有细胞膜的一个性能:最佳膜融合性能,在某些情况下还具有独特的细胞趋向性。并且来源于卵巢癌患者的外泌体在体外的聚乙二醇脂质体中,能够更好地发生免疫逃逸。通过进行体内外实验,利用纳米追踪分析和免疫印迹法分析外泌体,发现利用外泌体作为多柔比星的载体,相比单独多柔比星,心肌内皮细胞的外渗动力学相对缓慢;并且在存在免疫缺陷和具有免疫能力的小鼠中,对外泌体作为载体的多柔比星的容受剂量高于单纯的多柔比星。由此研究认为外泌体作为多柔比星载体,可以在不影响任何其他器官的情况下限制心脏毒性,增加乳腺癌和卵巢癌小鼠模型中多柔比星的治疗指数。卵巢癌作为妇科恶性疾病中最致死性病因,其中一部分的原因来自其化疗耐药,一些相关研究发现卵巢癌外泌体参与肿瘤化疗耐药的调节。研究发现铂类耐药的上皮性卵巢癌细胞中的外泌体可以转运一些自身化疗耐药的表型给铂类敏感细胞,增加耐药相关的EMT和sMAD4的突变,这证实了外泌体是上皮性卵巢癌铂类耐药的调节者。而且,研究发现卵巢癌的外泌体miR21可以通过向邻近基质细胞转运,使得卵巢癌细胞产生化疗耐药和侵袭陛更强的表型,APAFl是miR21的直接靶点,因此通过上调卵巢癌细胞的APAFl,是可以增加卵巢癌细胞对紫杉醇的敏感性。
发明内容
本发明一方面,制备并获得了脐带间充质干细胞外泌体。
进一步的,将化疗药物顺铂通过电击的方式导入到外泌体中,制备并获得了载药的外泌体。
更一步的,本发明提供了载药外泌体在制备用于治疗卵巢癌的药物中的用途。
更进一步的,本发明还通了特异性针对PI3K的单克隆抗体,该单克隆抗体能够特异性的抑制PI3K的信号通路,进而抑制卵巢癌细胞的增殖以及使其进行细胞凋亡。
更进一步的,本发明还提供了PI3K的单克隆抗体在制备治疗卵巢癌的药物中的用途。
进一步的,本发明的PI3K的单克隆抗体可抑制卵巢癌细胞p-AKT蛋白的表达,p-AKT是AKT的活化形式,AKT只有在活化状态时才有生物学特性,PI3K的单克隆抗体能通过抑制PI3K活性,进而减少AKT的活化,阻止其下游的分子发生生物学效应,导致细胞的增殖减少、凋亡增加,进而治疗卵巢癌。
进一步的,本发明的单克隆抗体能够通过增加化疗药物的敏感性来提高二者协同的治疗效果。
因此,进一步的,本发明还提供了PI3K的单克隆抗体在提高顺铂对卵巢癌细胞SK-OV-3药物敏感性中的用途。
本发明还提供了一种用于治疗卵巢癌的药物组合物,所述药物组合物是由PI3K的单克隆抗体和负载了顺铂的外泌体组成。
进一步的,所述的药物组合物中还含有药学上可接受的载体。
进一步的,载体是一种或多种选自以下的成分:粘合剂、填充剂、润滑剂和成对泡腾成分(effervescent couple)、芯吸剂(wicking agent)、助流剂、崩解剂和润湿剂。
在本发明的上下文中,“用于口服给药”指的是药物组合物在易于整个吞咽的固体剂型中,并且在给药前基本不溶解或混悬在水中。
适宜的崩解剂将是本领域技术人员已知的,非限制性的实例包括交联羧甲基纤维素钠、淀粉羟乙酸钠、交联聚乙烯吡咯烷酮、聚维酮、淀粉(例如玉米淀粉、预胶化淀粉)、低取代的羟丙基纤维素、海藻酸、海藻酸钠、三价磷酸钙、硫酸钙、羧甲基纤维素钙、微晶纤维素、粉状纤维素、二氧化硅胶体、多库酯钠、瓜尔胶、羟丙基纤维素、硅酸镁铝、甲基纤维素、聚克立林钾和聚乙烯吡咯烷酮。交联羧甲基纤维素钠是优选的。崩解剂可以单独使用或与其它物质组合。
有益效果
本发明提供了一种干细胞外泌体,所述外泌体通过负载化疗药物之后,能够有效的用于卵巢癌的治疗。将所述负载了化疗药物的外泌体与PI3K的单克隆抗体一起联用后,能够显著的提高化疗药物的敏感性,使得二者具有协同的显著的治疗优势,在应用前景上体现了较好的效果。
附图说明
图1外泌体不同的载药量对细胞存活率的影响
图2PI3K单克隆抗体对卵巢癌干细胞细胞存活率的影响
具体实施方式
本发明可通过后续对于本发明一些实施方案描述以及其中所包括的实施例的详细内容而更容易被了解。在进一步叙述本发明之前,应明了本发明不会被局限于所述特定实施方案中,因为这些实施方案必然是多样的。亦应明了本说明书中所使用的用语仅是为了阐述特定实施方案,而非作为限制,因为本发明的范围将会被仅仅界定在所附的权利要求中。
实施例1干细胞外泌体的制备
无菌取足月剖宫产胎儿脐带,在超净台内用含有双抗的PBS清洗脐带3遍,洗去残存血液,剔除脐带动、静脉和外包膜并将其剪碎至1mm3大小,均匀接种于一次性培养瓶中,使用含10%FBS的DMEM/F12的培养液置饱和湿度的37℃、5%CO2培养箱内培养。每隔3天更换1次培养液。培养约10d左右即有成纤维状细胞从组织中爬出,待细胞长至80%融合时,使用0.25%胰蛋白酶消化传代。将生长良好的P4代MSCs在37℃、5%CO2的条件下使用无血清培养液培养48h收集上清液。4℃条件下,300×g离心5min、2000×g离心20min、10000×g离心70min。随后将上清液移入高速离心管中,4℃条件下,1000000×g离心120min,得到沉淀物,在离心管中加满PBS,再次1000000×g离心120min,得到提纯的外泌体沉淀。将沉淀以PBS重悬,0.22μm滤膜过滤除菌,以BCA法检测其蛋白浓度,调整其蛋白浓度为1g/L,-80℃保存备用。
将分离纯化后的MSCs来源的exosomes与PE标记的鼠抗人CD73、CD44抗体室温避光孵育30min,用PBS洗涤1遍后,用1%的多聚甲醛重悬,上流式细胞仪检测分析。结果显示,干细胞外泌体高表达CD44(58.62%)和CD73(34.52%),说明分离制备得到了外泌体。
取20μL exosomes悬液滴于载样铜网光面上,室温条件下静置1min,用滤纸从一侧吸干液体,滴加20g/L的磷钨酸约20μL于载样铜网上,室温负染1min后滤纸吸干负染液,于白炽灯下烘烤约10min,透射电镜下观察发现,外泌体呈圆形或椭圆形的膜性小囊泡,直径80%分布在在21.3nm。
实施例2干细胞外泌体载药方法
将实施例1的外泌体均匀铺于10cm培养皿,常规培养,加入顺铂注射液,使其最终质量浓度为5g/L,添加到冷却的4mm电穿孔比色杯中,用Eppendorf Eporator在1000kV下电穿孔混合物5ms,37℃温育30分钟以恢复外泌体膜。30℃条件下400×g离心10min,去除沉淀,取上清液,15℃条件下10000×g离心2min,再次去除沉淀,取上清液,4℃条件下100000×g离心60min,得沉淀。将沉淀在0.5mLPBS中溶解,4℃条件下100000×g离心60min,反复洗涤沉淀,最后以0.5mLPBS溶解,4℃条件下保存备用。将载药外泌体分别进行倍稀释,使其中外泌体中的顺铂质量浓度分别为1、5、10、20、40、100μmol/L。
实施例3卵巢癌干细胞培养、传代、鉴定
人卵巢癌SK-OV-3细胞由含体积分数10%血清的DMEM/F12培养基常规培养。取对数生长期细胞以2.5g/L胰蛋白酶吹打制成单细胞悬液,计数细胞。以细胞密度2×107/L接种于SFM培养基中培养,取第5代悬浮细胞球和正常贴壁分化细胞,用胰蛋白酶消化获得单细胞悬液。使用PBS缓冲液洗涤细胞2次,调整细胞密度为2×109/L。分别取100μL细胞悬液加入FITC-CD44抗体10μL标记细胞,同时每种荧光抗体设同型对照,避光室温孵育15min,1000r//min离心5min,弃上清液,加入300μL的PBS,吹打均匀后上流式细胞仪检测。肿瘤细胞球和贴壁分化细胞中表达CD44的荧光强度分别为2.28±0.17、0.98±0.12,差异有显著性(P<0.05)。本发明通过无血清悬浮培养法富集制备了卵巢癌干细胞。
实施例4CCK-8检测筛选最佳外泌体载药量
取分离培养的卵巢癌干细胞,加入含20μg/L重组人表皮生长因子与20μg/L碱性成纤维细胞生长因子的RPMI1640培养基,制备单细胞悬液,以1×107/L的细胞浓度接种于96孔板,分别加入含顺铂质量浓度分别为1、5、10、20、40、100μmol/L载药外泌体的细胞培养基,以单纯细胞培养基培养的细胞为对照组,培养72h后,每孔加入10μLCCK-8溶液,继续在37℃、含体积分数5%CO2培养箱中培养2h,然后使用酶标仪在450nm波长处检测吸光度A值,计算细胞存活率,细胞存活率=实验组A值÷对照组A值×100%。结果如图1所示。
从图1的结果可以看出,培养72h后CCK-8检测发现,卵巢癌干细胞增殖活性受到不同程度的影响,随着药物浓度的增加,细胞存活率逐渐降低,当载药细胞外泌体中顺铂药物浓度为5μmol/L时,细胞存活率下降明显,因此实验选择顺铂载药浓度为5μmol/L的外泌体进行后续实验。
实施例5PI3K单克隆抗体P-6F17的获得
以重组人PI3K蛋白(ab125633,abcam)为免疫原,免疫8周龄雌性BALB/c小鼠,通过本领域常规的细胞融合方法并采用HAT和HT培养液进行培养筛选,筛选获得阳性杂交瘤细胞株,有限稀释法进行亚克隆,至阳性率为100%时,获得了特异性针对PI3K的单克隆抗体P-6F17。该抗体亚型是IgG1,具有较好的特异性,只与PI3K蛋白反应。通过表面离子共振亲和力鉴定发现其解离常数达到3.19×10-9M,特异性较好。其轻链可变区和重链可变区通过序列鉴定,分别如SEQ ID NO:1所示和SEQ ID NO:2所示。将所述抗体的杂交瘤细胞株进行腹水制备并进行抗体纯化备用。
实施例6PI3K单克隆抗体P-6F17对卵巢癌干细胞的影响
取分离培养的卵巢癌干细胞,加入含20μg/L重组人表皮生长因子与20μg/L碱性成纤维细胞生长因子的RPMI1640培养基,制备单细胞悬液,以1×107/L的细胞浓度接种于96孔板,分别加入含单抗P-6F17质量浓度分别为0、1、10、50、100、200μg/mL的单克隆抗体,以单纯细胞培养基培养的细胞为对照组,以浓度为100μg/mL的LY294002作为阳性对照,培养72h后,每孔加入10μLCCK-8溶液,继续在37℃、含体积分数5%CO2培养箱中培养2h,然后使用酶标仪在450nm波长处检测吸光度A值,计算细胞存活率,细胞存活率=实验组A值÷对照组A值×100%。
结果如图2所示,单抗P-6F17针对卵巢癌干细胞具有剂量依赖性的治疗效果,在浓度为200μg/mL的单克隆抗体条件下,细胞存活率达到了(34.39±
2.93)%,与阳性对照相比,具有相对较好的抑制效果。因此,选用200μg/mL的单抗P-6F17进行后续实验。
实施例7外泌体联合单抗P-6F17增敏实验
取分离培养的卵巢癌干细胞,加入含20μg/L重组人表皮生长因子与20μg/L碱性成纤维细胞生长因子的RPMI1640培养基,制备单细胞悬液,以1×107/L的细胞浓度接种于96孔板,分别采用如下各组进行处理:
实验组1:5μmol/L的顺铂;
实验组2:顺铂载药终浓度为5μmol/L的外泌体;
实验组3:200μg/mL的单克隆抗体P-6F17;
实验组4:顺铂载药浓度为5μmol/L的外泌体+200μg/mL的单克隆抗体P-6F17;
空白对照组:不加药,只加培养基;
作用细胞24h后,制成细胞悬液,离心后将细胞先用冷的PBS洗涤,75%乙醇于-20℃固定过夜,洗涤离心15min后加人含有10μg/mL PI和0.1%RNaseA的PBS液500μL,室温避光染色30min后用流式细胞仪测定细胞凋亡率。结果如表1所示。
表1各组对细胞凋亡率的影响结果
各实验组 | 凋亡率(%) |
实验组1 | 14.22±0.13* |
实验组2 | 18.39±0.24* |
实验组3 | 12.87±0.28* |
实验组4 | 34.28±0.31* |
空白对照组 | 1.32±0.12 |
(*表示与空白相比,差异显著,P<0.05)
从表1的结果可以看出,相同浓度的顺铂,采用外泌体负载后,对于细胞凋亡率具有一定的提高。但是,当单抗与负载顺铂的外泌体连用后,凋亡率得到显著的的提高。
收集经药物处理后的各组细胞,裂解细胞提取总蛋白,40μg蛋白经10%聚丙烯酞胺凝胶(SDS-PAGE电泳分离后转移至硝酸纤维素膜,室温封闭2h,一抗p-AKT 4℃敷育过夜,用含0.05%Tween20的TBS缓冲液漂洗3次,每次10min;加入相应的碱磷酶标记的二抗(1:500),室温lh,用5-嗅-4-氯-3-吲哚基磷酸酯/四唑氮蓝(BCIP/NBT/buffer)(l:l:50)试剂盒显色,β-actin作为内参,每个样本至少重复3次。结果如表2所示。
表2各组对细胞p-AKT表达的影响
各实验组 | p-AKT相对表达量(p-AKT/β-actin) |
实验组1 | 0.51±0.05* |
实验组2 | 0.50±0.07* |
实验组3 | 0.11±0.02* |
实验组4 | 0.09±0.01* |
空白对照组 | 0.54±0.08 |
(*表示与空白相比,差异显著,P<0.05)
从表2的结果可以看出,单克隆抗体可抑制卵巢癌细胞p-AKT蛋白的表达,p-AKT是AKT的活化形式,AKT只有在活化状态时才有生物学特性。因此,单克隆抗体能通过抑制PI3K,减少AKT的活化,阻止其下游的分子发生生物学效应,导致细胞的增殖减少、凋亡增加。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,但本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (8)
1.特异性针对PI3K的单克隆抗体P-6F17,其特征在于所述抗体的轻链可变区序列如SEQ ID NO:1所示,其重链可变区如SEQ ID NO:2所示。
2.特异性针对PI3K的单克隆抗体P-6F17通过抑制PI3K信号通路在制备治疗卵巢癌的药物中的用途。
3.负载化疗药物顺铂的脐带间充质干细胞外泌体和特异性针对PI3K的单克隆抗体P-6F17在制备治疗卵巢癌的药物组合物中的用途,其中特异性针对PI3K的单克隆抗体P-6F17的轻链可变区序列如SEQ ID NO:1所示,其重链可变区如SEQ ID NO:2所示。
4.特异性针对PI3K的单克隆抗体P-6F17通过抑制PI3K信号通路进而提高化疗药物顺铂药物对离体的卵巢癌细胞敏感性的药物中的用途;其中特异性针对PI3K的单克隆抗体P-6F17的轻链可变区序列如SEQ ID NO:1所示,其重链可变区如SEQ ID NO:2所示。
5.如权利要求2-4任一所述的用途,其中,所述卵巢癌是细胞SK-OV-3导致的。
6.如权利要求3所述的用途,其特征在于所述负载化疗药物顺铂的脐带间充质干细胞外泌体是通过将外泌体与顺铂注射液混合后,添加到冷却的电穿孔比色杯中,电穿孔将顺铂导入到外泌体中,37℃温育30分钟以恢复外泌体膜,进而制备得到负载化疗药物顺铂的脐带间充质干细胞外泌体。
7.如权利要求6所述的用途,其特征在于所述的药物组合物中还含有药学上可接受的载体。
8.一种用于治疗卵巢癌的药物组合物,所述组合物是由特异性针对PI3K的单克隆抗体P-6F17和负载了顺铂的脐带间充质干细胞外泌体组成;其中特异性针对PI3K的单克隆抗体P-6F17的轻链可变区序列如SEQ ID NO:1所示,其重链可变区如SEQ ID NO:2所示。
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