CN116585483A - 一种乏氧调节药物递送系统、其制备方法和应用 - Google Patents
一种乏氧调节药物递送系统、其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种乏氧调节药物递送系统、其制备方法和应用。本发明的乏氧调节药物递送系统可以实现肿瘤深部穿透、改善胶质瘤乏氧状态和靶向GSCs,并达到GSCs杀伤和抑制GBM生长的作用,为未来对GBM药物的开发和运用提供理论依据。
Description
技术领域
本发明属于纳米药物领域,具体涉及一种乏氧调节药物递送系统、其制备方法和应用。
背景技术
胶质瘤(Glioma,GBM)是一类恶性程度极高的脑部恶性肿瘤,其中位生存期仅仅15个月。在GBM的治疗中,常因为其手术切除不彻底和对化疗药物的耐受,造成肿瘤的复发,从而严重威胁患者的生存质量。在GBM中,存在一类数量很少的胶质瘤干细胞(Glioma stemcells,GSCs),约占1%-2%。这类细胞是GBM的起始细胞,具有去分化的特点。除此之外,GSCs在GBM细胞的恶性生长,迁移,耐药以及肿瘤的复发等方面都具有重要作用。因此,如何克服GBM中GSCs的生长是GBM治疗的重要方向。既往研究表明,GSCs的去分化状态主要依靠GBM的乏氧微环境来维持,在体外乏氧环境中培养肿瘤细胞后,肿瘤干性增强,肿瘤干细胞(Cancer stem cells,CSCs)标志物显著上调。因此将改善乏氧微环境作为辅助手段,可能促进GSCs对化学治疗的敏感性。除此之外,GSCs常常位于肿瘤的深层组织,化疗药物难以渗透和到达,对GSCs的杀伤作用十分有限,因此寻找新的药物载体来辅助化疗药物在肿瘤深层的渗透作用也至关重要。
针对肿瘤的乏氧微环境,BSA-MnO2载药体系能依赖于肿瘤酸性和过氧化氢富含的特点,来实现MnO2的催化产氧。同时,由于BSA中存在输水腔,可以将抗GSCs的化疗药物装载在其中,实现了以产氧为辅助手段的GSCs化疗增敏作用。但是,BSA-MnO2载药体系对GSCs的治疗仍然还存在一些局限性。例如,BSA-MnO2对血脑屏障(Blood brain barrier,BBB)的通透能力以及对肿瘤深部的穿透能力有限。BBB是由血管内皮细胞、星形胶质细胞轴突等组成了一道屏障结构,能有效阻止中枢神经系统外的毒素进入脑组织,对脑组织具有保护作用。但是,在中枢神经系统疾病中,BBB也能抑制外周药物对脑部的输入,从而大幅降低药物的治疗效果。因此如何增加BSA-MnO2载药体系对BBB的通透性在GBM的治疗中也至关重要。
小细胞外囊泡(Small extracellular veiscles,sEVs)是由各种细胞分泌的一类大小在50-150nm的具有脂质双分子层结构的细胞外囊泡。sEVs在肿瘤和正常组织细胞的信号传导和信息交流中具有重要作用。研究者发现,sEVs由于其BBB的通透能力,来源于中枢神经系统正常组织或病理组织的sEVs广泛分布于血液,尿液,脑脊液,唾液等体液中,并在中枢神经系统疾病的检测技术中发挥着重要作用。
因此,亟需提供一种具有良好透过血脑屏障的能力,能准确靶向中枢神经系统肿瘤,并能改善肿瘤乏氧微环境,产生MRI成像和肿瘤干细胞抑制的效果,对中枢神经系统肿瘤具有良好的抑制效果的药物递送系统,以克服现有技术中的缺陷,即传统载药体系虽然合成简便对肿瘤有一定的杀伤能力,但是其对肿瘤深层的穿透力以及在肿瘤干细胞抑制和对血脑屏障的通透性依然有限,因此在胶质瘤的治疗中也受到限制。
发明内容
因此,本发明的目的在于克服现有技术中的缺陷,提供一种乏氧调节药物递送系统、其制备方法和应用。本发明的乏氧调节药物递送系统可以实现肿瘤深部穿透、改善胶质瘤乏氧状态和靶向GSCs,并达到GSCs杀伤和抑制GBM生长的作用,为未来对GBM药物的开发和运用提供理论依据。
在阐述本发明内容之前,定义本文中所使用的术语如下:
术语“GBM”是指:Glioma,胶质瘤。
术语“GSCs”是指:Glioma stem cells,胶质瘤干细胞。
术语“CSCs”是指:Cancer stem cells,肿瘤干细胞。
术语“sEVs”是指:Small extracellular veiscles,小细胞外囊泡。
术语“BBB”是指:Blood brain barrier,血脑屏障。
术语“BMP”是指:同时络合了抗肿瘤药物PTC209和MnO2的BSA纳米颗粒。
术语“M1 sEVs”是指:M1巨噬细胞的小细胞外囊泡。
术语“BSA”是指:牛血清白蛋白。
术语“BSA-MnO2”是指:结合有MnO2的BSA纳米颗粒,简称“BM”。
术语“M1 sEVs-BMP”是指:包裹了M1巨噬细胞小细胞外囊泡的BMP纳米颗粒。
术语“DMSO”是指:二甲基亚砜。
术语“NR”是指:染料尼罗红。
术语“FBS”是指:胎牛血清。
术语“PTC209”是指:抗胶质瘤干细胞的小分子药PTC209。
术语“BP”是指:结合有PTC209的BSA纳米颗粒。
术语“PBS”是指:磷酸盐缓冲液。
术语“M1 sEVs-NR-BMP”是指:由M1巨噬细胞小细胞外囊泡修饰的并同时连接有PTC209和尼罗红染料的BSA纳米颗粒。
术语“NR-BMP”是指:同时连接有尼罗红和PTC209的纳米颗粒。
术语“BMI1”是指:B细胞特异性莫洛尼鼠白血病病毒插入位点1。
术语“Olig2”是指:少突胶质细胞系转录因子2。
术语“Sox2”是指:SRY盒基因2。
术语“GAPDH”是指:磷酸甘油醛脱氢酶。
为实现上述目的,本发明的第一方面提供了一种乏氧调节药物递送系统,所述乏氧调节药物递送系统包括:小细胞外囊泡和核心纳米颗粒,所述核心纳米颗粒是由抗肿瘤药物和BSA-MnO2结合而成的纳米颗粒;
优选地,所述小细胞外囊泡和所述核心纳米颗粒的结合方式选自以下一种或多种:电穿孔、共孵育、超声、膜挤出,更优选为超声或膜挤出,最优选为超声;和/或
优选地,所述抗肿瘤药物和所述BSA-MnO2的结合方式选自以下一种或多种:络合、静电吸附、化学键结合,更优选为络合或静电吸附,最优选为络合。
根据本发明第一方面的乏氧调节药物递送系统,其中,
所述小细胞外囊泡的来源选自以下一种或多种细胞:M1巨噬细胞、肿瘤细胞、神经干细胞,优选为M1巨噬细胞或肿瘤细胞,最优选为M1巨噬细胞;和/或
所述抗肿瘤药物为抗肿瘤干细胞的小分子脂溶性药物,优选为对肿瘤干细胞标志物有抑制作用的小分子脂溶性药物,最优选为肿瘤干细胞标志蛋白BMI1的小分子抑制剂PTC209。
根据本发明第一方面的乏氧调节药物递送系统,其中,
所述核心纳米颗粒的粒径为5~15nm,优选为6~13nm,更优选为7~12nm;
所述乏氧调节药物递送系统的粒径为50~200nm,优选为80~180nm,更优选为100~150nm;和/或
所述乏氧调节药物递送系统的载药形式选自以下一种或多种:电穿孔、共孵育、超声、膜挤出,优选为超声或膜挤出,最优选为超声。
根据本发明第一方面的乏氧调节药物递送系统,其中,
所述小细胞外囊泡和所述核心纳米颗粒的质量/体积比为50~400/1μg/mL,优选为200~400/1μg/mL,最优选为200/1μg/mL;和/或
所述抗肿瘤药物和所述BSA-MnO2的质量/体积比为0.2~2/1mg/mL,优选为0.5~1.5/1mg/mL,最优选为1/1mg/mL。
本发明的第二方面提供了制备第一方面所述的乏氧调节药物递送系统的方法,所述方法包括以下步骤:
(1)合成核心纳米颗粒;
(2)提取小细胞外囊泡;
(3)合成步骤(1)制备的核心纳米颗粒和步骤(2)制备的小细胞外囊泡,即得所述乏氧调节药物递送系统;
优选地,所述步骤(1)中,所述核心纳米颗粒的合成方法为水热法或化学键结合法,最优选为水热法;和/或
优选地,所述步骤(3)中,所述合成步骤(1)制备的核心纳米颗粒和步骤(2)制备的小细胞外囊泡的方法选自以下一种或多种:超声合成、共孵育,膜挤出,电穿孔,最优选为超声合成。
根据本发明第二方面的方法,其中,所述步骤(1)中包括以下步骤:在BSA溶液中依次加入MnCl2·4H2O和抗肿瘤药物,再加入碱性溶液调节pH值,恒温反应后即得所述核心纳米颗粒;
优选地,所述抗肿瘤药物和BSA-MnO2的质量/体积比为0.2~2/1mg/mL,更优选为0.5~1.5/1mg/mL,最优选为1/1mg/mL;
优选地,所述碱性溶液选自以下一种或多种:氢氧化钠溶液,氢氧化钾溶液,碳酸氢钠溶液,更优选为氢氧化钠溶液或氢氧化钾溶液,最优选为氢氧化钠溶液;
优选地,所述pH值为9~13,更优选为10~12,最优选为11;
优选地,所述恒温反应的温度为35~40℃,更优选为36~38℃,最优选为37℃;和/或
优选地,所述恒温反应的时间为1~4h,更优选为1.5~3h,最优选为2h。
根据本发明第二方面的方法,其中,
所述步骤(2)中包括以下步骤:提取小细胞上清液,依次经过离心、超速离心分离,纯化后即得所述小细胞外囊泡;和/或
所述步骤(3)中:所述小细胞外囊泡和所述核心纳米颗粒的质量/体积比为50~400/1μg/mL,优选为200~400/1μg/mL,最优选为200/1μg/mL;
所述超声的功率为20~50%,优选为30~45%,最优选为40%;和/或
所述超声的时间为10~30min,优选为10~20min,最优选为15min。
本发明的第三方面提供了第一方面所述的乏氧调节药物递送系统在制备造影剂中的应用;
优选地,所述造影剂为核磁共振造影剂,且不含有核磁成像功能的游离金属离子;和/或
优选地,所述造影剂通过乏氧调节药物递送系统中的MnO2在肿瘤酸性和过氧化氢条件下分解产生Mn2+从而产生核磁共振信号。
本发明的第四方面提供了第一方面所述的乏氧调节药物递送系统在制备用于抗肿瘤药物中的应用;
优选地,所述肿瘤选自以下一种或多种:胶质瘤原位瘤、皮下胶质瘤、脑转移瘤,更优选为胶质瘤原位瘤和/或皮下胶质瘤;
进一步优选地,所述胶质瘤为乏氧肿瘤。
根据本发明第四方面的应用,其中,所述抗肿瘤药物为肿瘤干细胞小分子抑制剂或肿瘤干细胞标志物siRNA,最优选为肿瘤干细胞抑制剂;
优选地,所述肿瘤干细胞选自以下一种或多种:胶质瘤干细胞、乳腺癌干细胞、白血病细胞,最优选为胶质瘤干细胞;和/或
优选地,所述肿瘤干细胞抑制剂具有抑制肿瘤细胞和/或肿瘤干细胞增殖活性的功能。
根据本发明的一个具体的实施方案,本发明第一方面提供了一种M1巨噬细胞小细胞外囊泡修饰的BSA-MnO2药物递送系统(M1 sEVs-BMP)。
第二方面提供了第一方面M1 sEVs-BMP的制备方法,其所述方法包括一下步骤:
(1)水热法合成BMP核心纳米颗粒。
(2)M1 sEVs的提取。
(3)超声合成M1 sEVs-BMP纳米颗粒。
根据本发明第二方面的制备方法,其中,所述步骤(1)包括以下步骤:
(a)将BSA溶解在生理盐水或超纯水中(60mg/mL BSA),并向其中加入MnCl2·4H2O(200mg/mL),随后逐滴加入溶解在DMSO中的脂溶性抗肿瘤药物PTC209(5μg/μL PTC209),150-200转/分搅拌均匀。
(b)在(a)体系中加入NaOH调至PH为11。
(c)将(b)中混合溶液置于37℃恒温摇床中以150-200转/分的速度反应2h。
根据本发明第二方面的制备方法,其中,所述步骤(2)包括以下步骤:
(A)以不同浓度(0,0.05,0.1,0.2,0.4,0.8mM)的Mn2+来促使Raw264.7细胞极化为抗肿瘤生长的M1型,优选地,Mn2+最终浓度为0.1mM。
(B)提取(A)处理后的细胞上清,并置于离心机中1500-2000g,20min离心。
(C)将(B)处理后的上清置于超速离心机中,120000-150000g,1.5-2h离心获得M1sEVs。
(D)对(C)中获得的M1 sEVs用200μL生理盐水稀释定量为1μg/μL,置于-80℃保存备用。
根据本发明第二方面的制备方法,其中,所述步骤(3)包括以下步骤:
(4)将(1)中获得的BMP与(2)中获得的M1 sEVs按比例混合(1ml BMP对应150-200μg M1 sEVs)。随后置于超声清洗仪中,以80-100W,40%-50%功率超声反应10-15min。
(5)将(4)中混合液放置于37℃恒温箱中保温1h。
本发明第三方面提供了一种造影剂,其原理主要由M1 sEVs-BMP中的MnO2在肿瘤酸性和过氧化氢环境中分解所生成的Mn2+产生。
优选地,所述造影剂为核磁共振(MRI)造影剂.
本发明第四方面提供了第一方面所述的M1巨噬细胞小细胞外囊泡修饰的BSA-MnO2药物递送系统(M1 sEVs-BMP)和/或按照第二方面所述的制备方法而制得的M1 sEVs-BMP用于治疗GBM的药物中的应用;优选地,所述胶质母细胞瘤(GBM)为乏氧肿瘤。
本发明第五方面提供了肿瘤干细胞抑制剂,优选地,肿瘤干细胞为胶质母细胞瘤干细胞。所述肿瘤干细胞抑制剂抑制剂包括第一方面所述的M1巨噬细胞小细胞外囊泡修饰的BSA-MnO2药物递送系统(M1 sEVs-BMP)和/或按照第二方面所述的制备方法而制得的M1sEVs-BMP。
由于sEVs能穿透肿瘤组织并到达其深处部位,因此sEVs修饰的纳米颗粒能有效克服以往纳米材料的穿透性问题。因此,sEVs为肿瘤深部GSCs的治疗提供了纳米载药平台。
外泌体是一种能实现血脑屏障跨越和肿瘤深层穿透以及CSCs靶向的良好载体。M1巨噬细胞外泌体具有将促进肿瘤生长M2型巨噬细胞转化为抑制肿瘤生长M1型巨噬细胞的能力。由于BSA-MnO2载药体系抑制肿瘤乏氧微环境后,能促使肿瘤干细胞CD47表达下调,抑制“不要吃我”信号,从而促进M1巨噬细胞对CSCs的吞噬作用。因此,本发明结合了M1巨噬细胞外泌体和BSA-MnO2载药体系并将抗GSCs化疗药物PTC209包载在其中,成功实现了BBB通透、GBM深层穿透的功能。除此之外,还实现了以改善GBM乏氧为中心的GSCs化疗增敏以及M1巨噬细胞对GSCs的吞噬作用。
本发明所述的M1 sEVs-BMP纳米颗粒能被胶质瘤细胞和胶质瘤干细胞所摄取,并能很好透过血脑屏障能力,在体内实验和体外实验均表现出了对胶质瘤的强效杀伤能力。M1 sEVs-BMP通过抑制肿瘤乏氧来增强对肿瘤起始细胞的杀伤作用,在胶质瘤的治疗中具有广泛前景。
本发明涉及生物医药技术领域,具体而言,涉及一种抑制肿瘤乏氧状态、抑制肿瘤细胞和肿瘤干细胞增殖活性的纳米颗粒及其在胶质瘤治疗上的应用。
本发明的乏氧调节药物递送系统可以具有但不限于以下有益效果:
1、本发明以BSA做为载体,方法快速简便,成本低,稳定可靠,容易扩大生产。以BSA为载体克服了输水药物在水中溶解度低的问题,方便静脉给药。
2、本发明所获得的纳米颗粒具有较高的载药率,化疗药物装载后性质稳定,保存时间长,分散性良好。由于是天然的生物蛋白,因此在生物体内生物相容性良好,副作用较少。
3、本发明所获得的纳米颗粒具有透过血脑屏障的能力,能准确靶向中枢神经系统肿瘤,并能改善肿瘤乏氧微环境,产生MRI成像和肿瘤干细胞抑制的效果。对中枢神经系统肿瘤具有良好的抑制效果。
4、本发明所获得的纳米颗粒具有MRI成像的功能,可以在治疗胶质瘤的同时监测肿瘤大小的变化,达到诊疗一体化的目的。
附图说明
以下,结合附图来详细说明本发明的实施方案,其中:
图1示出了实施例1中M1巨噬细胞小细胞外囊泡以及M1 sEVs-BMP的形貌图。
图2示出了实施例2中M1 sEVs-BMP对胶质瘤细胞株GL261的体外杀伤作用及干细胞标志物抑制作用,体外GL261细胞活性采用CCK8试剂盒(索莱宝)通过酶标仪(ThermoScientific)450nm紫外吸收出测得;其中,图2a示出了M1 sEVs-BMP对胶质瘤细胞株GL261的体外杀伤作用;图2b示出了M1 sEVs-BMP对胶质瘤干细胞标志物BMI1,Sox2,Olig2的抑制作用。
图3示出了实施例3中胶质瘤细胞对BMP以及M1 sEVs-BMP的摄取能力。BMP以及M1sEVs-BMP均采用尼罗红(NR)染色,并通过流式细胞术测得NR进入到细胞中的荧光强度。
图4示出了实施例4中BMP以及M1 sEVs-BMP在体外对BBB的通透性。体外血脑屏障(BBB)模型采用小鼠bEnd.3脑微血管内皮细胞并通过0.4微米迁移小室(transwell)共培养获得。BMP以及M1 sEVs-BMP均采用尼罗红(NR)染色,并通过流式细胞术(BD Accuri C6)测得NR跨越BBB的荧光强度以及被下室肿瘤细胞摄取的荧光强度;其中,图4a示出了BBB模型中,M1 sEVs-BMP跨越BBB后下室中M1 sEVs-BMP的荧光强度;图4b示出了BBB模型中,M1sEVs-BMP跨越BBB后下室后,被下室胶质瘤细胞摄取的荧光强度。
图5示出了实施例5中M1 sEVs-BMP在体外的MRI成像功能。
图6示出了实施例6中胶质瘤原位荷瘤小鼠在尾静脉注射M1sEVs-BMP后对肿瘤生长的抑制能力。肿瘤大小的定量,主要使用活体成像仪并通过腹注射萤光虫素钠盐后与Luc(+)GL261反应后的生物发光测得。
图7示出了实施例7中M1 sEVs-BMP对荷瘤小鼠肿瘤中GSCs标志物的抑制能力;其中,图7a示出了M1 sEVs-BMP对胶质瘤干细胞标志物BMI1的抑制能力;图7b示出了M1 sEVs-BMP对胶质瘤干细胞标志物Olig2的抑制能力。
图8示出了M1 sEVs-BMP中MnO2在体外和体内产生氧气的能力,证明了M1 sEVs-BMP能有效缓解胶质瘤的乏氧状态;其中,图8a示出了体外BM,BMP和M1 sEVs-BMP在不同微环境中产生氧气的速率;图8a示出了M1 sEVs-BMP在体内胶质瘤中产生氧气并抑制乏氧标志物HIF-1ɑ表达的能力,标尺:100微米。
具体实施方式
下面通过具体的实施例进一步说明本发明,但是,应当理解为,这些实施例仅仅是用于更详细具体地说明之用,而不应理解为用于以任何形式限制本发明。
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的,但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚,在上下文中,如果未特别说明,本发明所用材料和操作方法是本领域公知的。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
除非特别指明,以下实施例中所用的巨噬细胞系Raw264.7和胶质瘤细胞系Luc(+)GL261均来自于国家纳米科学中心。
除非特别指明,以下实施例中M1 sEVs-BMP的合成以及合成后的纳米颗粒均重悬于生理盐水中。
除非特别指明,以下实施例中GL261细胞株培养基均采用DMEM/F12+10%FBS+1%双抗。Raw264.7和bEnd.3细胞均采用DMEM+10%FBS+1%双抗进行培养。
除非特别指明,所有荷瘤小鼠均采用Luc(+)GL261细胞经脑立体定位仪(ThermoScientific)原位接种成瘤。
以下实施例中使用的试剂和仪器如下:
试剂:
牛血清白蛋白(0332)购自于拜尔迪生物科技有限公司;氯化锰(G09Z005)购自阿法爱莎公司;抗肿瘤药物PTC209(HY-15888)购自MCE公司;DMEM(C11995500BT),DMEM/F12(C11330500BT),双抗(15140122)均购自Gibco公司;二甲基亚砜(D8371)购自索莱宝有限公司;小鼠Luc-GL261,小鼠bEnd.3,小鼠Raw264.7细胞系均来自于国家纳米科学中心;CCK8(CK04)试剂盒购置于日本同仁公司;尼罗红(HY-D0718)购置于MCE公司;氢氧化钠(1310-73-2)购置于索莱宝公司。
仪器:
生物透射电镜购自日本日立科技有限公司,型号HT7700;纳米颗粒跟踪分析仪购自英国马尔文仪器有限公司,型号NS300;单光子共聚焦显微镜购自德国蔡司有限公司,型号Zeiss710;流失细胞仪购自美国BD公司,型号C6;酶标仪购自美国分子仪器(上海)有限公司,型号Molecular Devices13。
实施例1
本实施例主要说明M1 sEVs的提取、M1 sEVs-BMP的合成及其形貌表征。
首先,M1巨噬细胞在0.1mM的锰离子环境中培养24h,随后提取细胞上清液,2000g,20min离心去除细胞和细胞碎片以及大粒径的囊泡。将处理后的上清通过0.22μm的滤膜进一步纯化。随后,上清再经过2次超速离心(150000g,1.5h)分离并纯化得到M1 sEVs。
其次,BMP核心纳米通过水热一步法合成。2mL的BSA(60mg/mL)与100μl的MnCl2(12.6mg/mL)混合后,加入DMSO溶解的PTC209药物(5mg/mL)并使用NaOH调节pH至11。经过37℃,2h反应后得到棕色的BMP核心纳米颗粒。
M1 sEVs-BMP的合成主要通过超声的方法进行。将200μg的M1 sEVs与1mL的BMP混合后,在超声清洗仪上进行40%功率,15min的反应,反应完成后将混合液放入37℃稳定1h等待膜结构恢复。
图1出示了实施例1中M1 sEVs-BMP的电镜结构,证明了M1sEVs-BMP的成功合成。
实施例2
本实施例主要说明M1 sEVs-BMP对胶质瘤细胞株GL261的体外杀伤作用及干细胞标志物抑制作用。
GL261以1万个细胞/孔的密度接种于96孔板中,随后向孔内加入不同浓度PTC209的纳米颗粒(分为PBS,BM,BP,BMP,M1 sEVs-BMP组)。待其培养24h后,各孔加入终浓度为10%的CCK8试剂,反应2-4h后通过酶标仪测得450nm处的紫外吸收。
为了检测M1 sEVs-BMP对GL261细胞株中干细胞的杀伤能力,GL261以40万个细胞/孔的密度接种于6孔板中,过夜培养等待期完全贴壁后,加入终浓度为2Μm PTC209的纳米颗粒(分为PBS,BM,BP,BMP,M1sEVs-BMP组),24h的培养后,将GL261刮下,裂解,煮蛋白并与适量Loading buffer混合。BCA蛋白定量后,使用WB技术检测各组药物对GL261中干细胞标志物的抑制作用。
图2a出示了实施例2中M1 sEVs-BMP对GL261胶质瘤细胞系的杀伤作用,图2b出示了实施例2中M1 sEVs-BMP对胶质瘤干细胞标志物BMI1,Sox2,Olig2的抑制作用。说明M1sEVs-BMP能有效抑制GL261胶质瘤细胞的生长并能抑制其干细胞的表达。
实施例3
本实施例主要说明胶质瘤细胞对BMP以及M1 sEVs-BMP的摄取能力。
首先,GL261以30万个细胞/孔的密度接种于6孔板中。BMP以及M1 sEVs-BMP均通过尼罗红染色,尼罗红在BSA的包载同PTC209。随后,以1μM的NR浓度加入细胞中,培养4h后,通过流式细胞学检测GL261细胞中NR的荧光强度。
图3出示了实施例3中被NR染色的M1 sEVs-BMP纳米颗粒被胶质瘤细胞摄取的能力。证明在包裹M1 sEVs后的纳米颗粒被细胞的摄取能力更强。
实施例4
本实施例主要说明M1 sEVs-BMP在体外对BBB的通透性。
首先,建立体外BBB模型。bEnd.3细胞以2万个细胞/孔接种于上层0.4μm Traswell小室中,上下室均需添加培养基以促进细胞生长。每天检测跨膜电阻,当跨膜电阻大于150ΩcM-2时说明BBB模型构建成功。
随后,将构建成功的BBB小室置于种植有GSCs的培养板上层。在上室中加入终浓度为10μM的NR,经4h共培养后,取下室中的培养液于酶标仪检测488/570nm处的NR荧光强度,取下室GSCs细胞通过流式细胞学分析摄取NR的荧光强度。
图4a和图4b出示了M1 sEVs-BMP对体外BBB模型的通透性,经酶标仪和流式细胞仪检测,下室M1 sEVs-NR-BMP较NR-BMP具有更强的荧光信号且下室肿瘤干细胞对M1 sEVs-NR-BMP的摄取能力更强。证明M1sEVs包裹后的M1 sEVs-BMP纳米颗粒具有更强的BBB透过能力。
实施例5
本实施例主要说明M1 sEVs-BMP在体外的MRI成像功能。
由于M1 sEVs-BMP中的MnO2在肿瘤酸性和过氧化氢条件下分解产生Mn2+从而产生MRI信号。我们在体外模拟了酸性和过氧化氢的肿瘤微环境,其具体分组为BM+pH7.4,BM+pH6.5,BM+pH6.5+H2O2,BMP+pH6.5+H2O2,M1 sEVs-BMP+pH6.5+H2O2。随后将各组材料分别稀释成终浓度为0.12,0.24,0.48,0.96,1.92mM的溶液并置于100μL的EP管中进行3.0T的MRI成像。
图5出示了实施例5中,M1 sEVs-BMP纳米颗粒随浓度梯度的升高MIR成像的能力越来越强。
实施例6
本实施例主要说明胶质瘤原位荷瘤小鼠在尾静脉注射M1 sEVs-BMP后对肿瘤生长的抑制能力。
首先,构建体内胶质瘤原位瘤模型。所有的C57 BL/6雄性小鼠(6-8周龄,15-20g)均购置于北京维通利华公司。所有的动物实验均遵循国家纳米科学中心动物伦理条例并获得动物伦理委员会批准。50万个Luc(+)GL261细胞被接种于小鼠颅内。其具体方法为:1%戊巴比妥麻醉小鼠后,剃去头部毛发并固定于脑立体定位仪上,碘伏消毒后,剪刀剪开皮肤暴露前囟,在前囟右侧2mm,后1mm处使用磨钻钻开颅骨并不伤及硬脑膜,随后采用微量注射器将肿瘤细胞种植于3mm深的脑实质部位。随后,骨蜡封闭缺口并消毒缝合,将小鼠置于电热毯上待其苏醒。
其次,尾静脉注射纳米颗粒。实验分为PBS,BM,BP,BMP,M1sEVs-BMP组,以10mg/mL的PTC209终浓度尾静脉隔天注射,并于对应时间活体成像检测肿瘤大小。
图6出示了M1 sEVs-BMP在体内模型中对胶质瘤的生长抑制作用,其作用优于其余各组。
实施例7
本实施例主要说明M1 sEVs-BMP对荷瘤小鼠肿瘤中GSCs标志物的抑制能力,以及改善肿瘤乏氧状态后对GSCs的杀伤增强作用。
将实施例6中治疗后的小鼠取出脑组织,并多聚甲醛固定24h,随后进行免疫组化染色分析肿瘤干细胞标志物的表达。
图7a和图7b出示了体内胶质瘤干细胞标志物BMI1和Olig2的表达。说明M1 sEVs-BMP纳米颗粒能显著抑制小鼠胶质瘤干细胞的生长。且在氧气产生后,对GSCs的杀伤作用更强。
实施例8
本实施例主要说明M1 sEVs-BMP中MnO2的体外产生氧气的能力,以及在体内胶质瘤中抑制乏氧标志物HIF-1ɑ的作用。
体外实验,取BM,BMP和M1 sEVs-BMP各2mL加入试管中,随后用1%稀盐酸将其pH值调值6.5并加入10-4M的H2O2,随后使用便携式测氧仪(泰州雷磁仪器有限公司)每10秒检测氧气的产生量。
体内实验,将实施例6中治疗后的小鼠取出脑组织,并多聚甲醛固定24h,随后进行免疫组化染色分析HIF-1ɑ的表达。
图8a出示了体外BM,BMP和M1 sEVs-BMP在不同微环境中产生氧气的速率。
图8b出示了M1 sEVs-BMP在体内胶质瘤中产生氧气并抑制乏氧标志物HIF-1ɑ表达的能力,标尺:100微米。
图8a和图8b出示了M1 sEVs-BMP中MnO2在体外和体内产生氧气的能力,证明了M1sEVs-BMP能有效缓解胶质瘤的乏氧状态。
尽管本发明已进行了一定程度的描述,明显地,在不脱离本发明的精神和范围的条件下,可进行各个条件的适当变化。可以理解,本发明不限于所述实施方案,而归于权利要求的范围,其包括所述每个因素的等同替换。
Claims (10)
1.一种乏氧调节药物递送系统,其特征在于,所述乏氧调节药物递送系统包括:小细胞外囊泡和核心纳米颗粒,所述核心纳米颗粒是由抗肿瘤药物和BSA-MnO2结合而成的纳米颗粒;
优选地,所述小细胞外囊泡和所述核心纳米颗粒的结合方式选自以下一种或多种:电穿孔、共孵育、超声、膜挤出,更优选为超声或膜挤出,最优选为超声;和/或
优选地,所述抗肿瘤药物和所述BSA-MnO2的结合方式选自以下一种或多种:络合、静电吸附、化学键结合,更优选为络合或静电吸附,最优选为络合。
2.根据权利要求1所述的乏氧调节药物递送系统,其特征在于:
所述小细胞外囊泡的来源选自以下一种或多种细胞:M1巨噬细胞、肿瘤细胞、神经干细胞,优选为M1巨噬细胞或肿瘤细胞,最优选为M1巨噬细胞;和/或
所述抗肿瘤药物为抗肿瘤干细胞的小分子脂溶性药物,优选为对肿瘤干细胞标志物有抑制作用的小分子脂溶性药物,最优选为肿瘤干细胞标志蛋白BMI1的小分子抑制剂PTC209。
3.根据权利要求1或2所述的乏氧调节药物递送系统,其特征在于:
所述核心纳米颗粒的粒径为5~15nm,优选为6~13nm,更优选为7~12nm;
所述乏氧调节药物递送系统的粒径为50~200nm,优选为80~180nm,更优选为100~150nm;和/或
所述乏氧调节药物递送系统的载药形式选自以下一种或多种:电穿孔、共孵育、超声、膜挤出,优选为超声或膜挤出,最优选为超声。
4.根据权利要求1至3中任一项所述的乏氧调节药物递送系统,其特征在于:
所述小细胞外囊泡和所述核心纳米颗粒的质量/体积比为50~400/1μg/mL,优选为200~400/1μg/mL,最优选为200/1μg/mL;和/或
所述抗肿瘤药物和所述BSA-MnO2的质量/体积比为0.2~2/1mg/mL,优选为0.5~1.5/1mg/mL,最优选为1/1mg/mL。
5.制备权利要求1至4中任一项所述的乏氧调节药物递送系统的方法,其特征在于,所述方法包括以下步骤:
(1)合成核心纳米颗粒;
(2)提取小细胞外囊泡;
(3)合成步骤(1)制备的核心纳米颗粒和步骤(2)制备的小细胞外囊泡,即得所述乏氧调节药物递送系统;
优选地,所述步骤(1)中,所述核心纳米颗粒的合成方法为水热法或化学键结合法,最优选为水热法;和/或
优选地,所述步骤(3)中,所述合成步骤(1)制备的核心纳米颗粒和步骤(2)制备的小细胞外囊泡的方法选自以下一种或多种:超声合成、共孵育,膜挤出,电穿孔,最优选为超声合成。
6.根据权利要求5所述的方法,其特征在于,所述步骤(1)中包括以下步骤:在BSA溶液中依次加入MnCl2·4H2O和抗肿瘤药物,再加入碱性溶液调节pH值,恒温反应后即得所述核心纳米颗粒;
优选地,所述抗肿瘤药物和BSA-MnO2的质量/体积比为0.2~2/1mg/mL,更优选为0.5~1.5/1mg/mL,最优选为1/1mg/mL;
优选地,所述碱性溶液选自以下一种或多种:氢氧化钠溶液,氢氧化钾溶液,碳酸氢钠溶液,更优选为氢氧化钠溶液或氢氧化钾溶液,最优选为氢氧化钠溶液;
优选地,所述pH值为9~13,更优选为10~12,最优选为11;
优选地,所述恒温反应的温度为35~40℃,更优选为36~38℃,最优选为37℃;和/或
优选地,所述恒温反应的时间为1~4h,更优选为1.5~3h,最优选为2h。
7.根据权利要求5或6所述的方法,其特征在于,
所述步骤(2)中包括以下步骤:提取小细胞上清液,依次经过离心、超速离心分离,纯化后即得所述小细胞外囊泡;和/或
所述步骤(3)中:所述小细胞外囊泡和所述核心纳米颗粒的质量/体积比为50~400/1μg/mL,优选为200~400/1μg/mL,最优选为200/1μg/mL;
所述超声的功率为20~50%,优选为30~45%,最优选为40%;和/或
所述超声的时间为10~30min,优选为10~20min,最优选为15min。
8.权利要求1至4中任一项所述的乏氧调节药物递送系统在制备造影剂中的应用;
优选地,所述造影剂为核磁共振造影剂,且不含有核磁成像功能的游离金属离子;和/或
优选地,所述造影剂通过乏氧调节药物递送系统中的MnO2在肿瘤酸性和过氧化氢条件下分解产生Mn2+从而产生核磁共振信号。
9.权利要求1至4中任一项所述的乏氧调节药物递送系统在制备用于抗肿瘤药物中的应用;
优选地,所述肿瘤选自以下一种或多种:胶质瘤原位瘤、皮下胶质瘤、脑转移瘤,更优选为胶质瘤原位瘤和/或皮下胶质瘤;
进一步优选地,所述胶质瘤为乏氧肿瘤。
10.根据权利要求9所述的应用,其特征在于,所述抗肿瘤药物为肿瘤干细胞小分子抑制剂或肿瘤干细胞标志物siRNA,最优选为肿瘤干细胞抑制剂;
优选地,所述肿瘤干细胞选自以下一种或多种:胶质瘤干细胞、乳腺癌干细胞、白血病细胞,最优选为胶质瘤干细胞;和/或
优选地,所述肿瘤干细胞抑制剂具有抑制肿瘤细胞和/或肿瘤干细胞增殖活性的功能。
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