CN115089723A - 一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒及其制备方法和应用 - Google Patents
一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒及其制备方法和应用 Download PDFInfo
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- CN115089723A CN115089723A CN202210750642.8A CN202210750642A CN115089723A CN 115089723 A CN115089723 A CN 115089723A CN 202210750642 A CN202210750642 A CN 202210750642A CN 115089723 A CN115089723 A CN 115089723A
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Abstract
本发明提供了一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒及其制备方法和应用。所述锰基纳米颗粒含有三价锰离子和二硫键,具有良好的水分散性、生物相容性、肿瘤选择性富集、氧化还原响应性,对谷胱甘肽和过氧化氢十分敏感,能通过三种途径(高效消耗肿瘤细胞内的GSH、H2O2,以及激活cGAS‑STING通路)同时诱发铁死亡,进一步提升了药物疗效,为铁死亡纳米诊疗制剂提供了一种新的选择。
Description
技术领域
本发明属于肿瘤治疗技术领域。更具体地,涉及一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒及其制备方法和应用。
背景技术
铁死亡(Ferroptosis)是一种新型的细胞死亡模式,其发生依赖于细胞中铁的存在和脂质的过氧化,与凋亡、坏死和自噬有较大差别。自Stockwell于2012年提出并命名铁死亡以来,就有众多的国内外学者热衷于对铁死亡进行探索,已有研究发现在肝癌、乳腺癌、胰腺癌和前列腺癌中,铁死亡可以抑制肿瘤细胞的增殖,因此诱导铁死亡有望成为肿瘤治疗的新方式。
随着国内外大量关于铁死亡的研究开展,众多引发铁死亡的新机制不断被发现,主要为以下三种机制:1)通过二硫键-硫醇交换反应以及金属离子与肿瘤细胞内富集的谷胱甘肽(glutathioMe,GSH)之间的氧化还原反应,消耗掉肿瘤细胞内的GSH,从而抑制谷胱甘肽过氧化物酶4(GlutathioMe Peroxidase 4,GPX4)的活性,细胞抗过氧化能力降低,脂质活性氧(Reactive OxygeM Species,ROS)堆积,造成肿瘤细胞发生铁死亡;2)MM2+与肿瘤细胞内富集的H2O2发生类芬顿(FeMtoM-like)反应生成羟基自由基(·OH),·OH作为ROS的一种,可和细胞内脂质体反应生成脂质过氧化物,从而引起细胞内脂质活性氧堆积,造成肿瘤细胞发生铁死亡;3)MM2+激活细胞的cGAS-STING(the cyclic GMP-AMP syMthase/stimulator of iMterferoM geMes pathway)通路,活化CD8+T细胞,释放干扰素γ(IFNγ)下调SLC7A11(谷氨酸胱氨酸反转运蛋白System Xc -的亚基)的表达,从而抑制肿瘤细胞对胱氨酸的摄取,阻碍GSH的吸收,导致GPX4活性降低,细胞抗过氧化能力降低,脂质活性氧堆积,造成肿瘤细胞发生铁死亡。
但目前的铁死亡诱导剂大多只能通过单一途径去诱发铁死亡,导致诱导剂对肿瘤的疗效不佳、易产生耐受,如现有技术公开了一种尺寸小于30Mm的四氧化三铁纳米颗粒,仅能通过消耗GSH以抑制GPX4的活性这单一途径去诱发铁死亡,导致疗效不佳且易产生耐受。因此,寻找一种能同时通过以上三种途径去发挥功效的铁死亡诱导剂,对于肿瘤治疗具有相当的必要性。
发明内容
本发明针对现有铁死亡诱导剂大多只能通过单一途径去诱发铁死亡的技术问题,旨在提供一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒,同时通过三种途径诱发肿瘤细胞的铁死亡,进一步提升铁死亡诱导剂针对肿瘤的疗效,为铁死亡纳米诊疗制剂提供了一种新的选择。
本发明的另一目的是提供上述锰基纳米颗粒的制备方法。
本发明的又一目的是提供上述锰基纳米颗粒在制备抗肿瘤药物中的应用。
本发明上述目的通过以下技术方案实现:
本发明提供了一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒,所述锰基纳米颗粒含有三价锰离子和二硫键。
本发明研究发现,含有三价锰离子和二硫键的纳米颗粒,对谷胱甘肽和过氧化氢十分敏感,能通过三种途径同时诱发铁死亡,克服了现有铁死亡诱导剂只能通过单一途径诱发铁死亡,进而导致疗效不佳且易产生耐受的问题。其中,所述谷胱甘肽和过氧化氢敏感的锰基纳米颗粒诱导铁死亡的三种途径如图1所示,分别为:1)通过二硫键-硫醇交换反应消耗GSH(~SS~+GSH→2HS~+GSSH),通过金属离子与肿瘤细胞内富集的GSH之间的氧化还原反应消耗GSH(MM3++GSH→MM2++GSSH),从而抑制GPX4的活性,细胞抗过氧化能力降低,脂质ROS堆积,造成肿瘤细胞发生铁死亡;2)前述生成的MM2+与肿瘤细胞内富集的H2O2发生类芬顿(FeMtoM-like)反应生成·OH(MM2++H2O2→MM3++·OH+OH-),·OH和细胞内脂质体反应生成脂质过氧化物,从而引起细胞内脂质活性氧堆积,造成肿瘤细胞发生铁死亡;3)前述生成的MM2+还能有效激活细胞的cGAS-STING通路,活化CD8+T细胞,释放IFNγ下调SLC7A11的表达,从而抑制肿瘤细胞对胱氨酸的摄取,阻碍GSH的吸收,导致GPX4活性降低,细胞抗过氧化能力降低,脂质活性氧堆积,造成肿瘤细胞发生铁死亡。
本发明还提供了一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒的制备方法,该方法包括如下步骤:
S1.三价锰盐与二硫化物通过配位反应得到MMMⅢM-SS;
S2.甲氧基聚乙二醇与MMMⅢM-SS通过酯化反应得到所述锰基纳米颗粒MMMⅢM-SSNPs。
本发明方法制得的锰基纳米颗粒MMMⅢM-SS NPs具有良好的水分散性、生物相容性、肿瘤选择性富集、氧化还原响应性,能通过高效消耗肿瘤细胞内的GSH和H2O2、激活cGAS-STING通路等方式诱导肿瘤细胞铁死亡,且对cGAS-STING通路的激活还能显着促进宿主抗原递呈细胞对肿瘤抗原的递呈能力,促进细胞毒性T细胞在肿瘤组织内的浸润和肿瘤特异性杀伤,放大免疫驱动的铁死亡。此外,所述锰基纳米颗粒在肿瘤细胞中生成的MM2+具有磁共振成像探针的功能,可进行肿瘤成像,提高肿瘤部位T1磁共振成像效果,实现体内药物输送及治疗的无创监测,以及肿瘤精准可控的可视化治疗,改善非特异性器官及组织分布,提高生物利用度和治疗效果,实现特异性治疗,成为诊疗一体化的纳米药物体系,为肿瘤的诊疗提供了一种新的选择。
由于细胞内铁含量较低,简单的基因或分子试剂可能无法有效地提高芬顿反应的效率,不能产生大量羟基自由基去引起脂质过氧化物堆积,导致诱发铁死亡的效率较低,且基因和小分子特异性较弱、副作用大等缺陷也限制了其在临床上的应用。基于此,本发明将铁死亡诱导剂与无载体纳米药物递送系统有机结合在一起,将铁死亡诱导剂制备成无载体纳米颗粒,使其具有无载体纳米药物毒副作用小、载药能力高、特异性高等优点,避免非活性物质的添加带来潜在副作用与未知风险。
甲氧基聚乙二醇在该制备工艺中,作为改性剂、反应终止剂、亲水涂层分子,将疏水的MMMⅢM-SS改性成为亲水材料,且其自身含有的羟基可与MMMⅢM-SS中的羧基发生酯化反应。
优选地,所述三价锰盐包括三价醋酸锰、三价硫酸锰、三价磷酸锰中的一种或几种。
优选地,所述二硫化物包括3,3'-二硫代二丙酸、2,2'-二硫代二丙酸、3,3'-二硫代二丙酸、4,4'-二硫代二丁酸中的一种或几种。
优选地,所述三价锰盐与二硫化物的摩尔比为1~2:1。
最优选地,所述三价锰盐与二硫化物的摩尔比为1:1。
优选地,S1为:三价锰盐与二硫化物在有机溶剂中超声、加热,并经后处理得到MMMⅢM-SS。
进一步优选地,所述有机溶剂包括N,N-二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)、四氢呋喃(THF)中的一种或几种。
进一步优选地,所述超声的时间为30~60miM。
进一步优选地,所述加热为在110~150℃下加热24~48h。
进一步优选地,所述后处理为:将反应体系冷却至20~30℃,再在6000~10000rpm下离心5~20miM,最后用甲醇和DMF洗涤直至上清液无色。
优选地,所述甲氧基聚乙二醇与MMMⅢM-SS的摩尔比为4~8:1,最优选为5:1。
优选地,所述甲氧基聚乙二醇的分子量为1000~10000,最优选为5000(即mPEG-OHM5KM)。
优选地,S2为:甲氧基聚乙二醇、MMMⅢM-SS、活化剂、催化剂在有机溶剂中避光加热,并经后处理得到所述锰基纳米颗粒。
进一步优选地,MMMⅢM-SS、活化剂、催化剂的摩尔比为1:1~2:1~2,最优选为1:1:1。
进一步优选地,所述活化剂包括但不限于EDC。
进一步优选地,所述催化剂包括但不限于4-二甲氨基吡啶(DMAP)。
进一步优选地,所述有机溶剂包括四氢呋喃(THF)、二甲基亚砜(DMSO)、N,N-二甲基甲酰胺(DMF)中的一种或几种。
进一步优选地,所述避光加热为在避光环境中、40~50℃下加热24~48h。
进一步优选地,所述后处理为:去除有机溶剂后,转移至截留分子量(MWCO)为7000Da的透析袋中,用蒸馏水透析24~72h,且透析过程中,每4~8h更换一次蒸馏水,最后冷冻干燥。
上述方法制备得到的锰基纳米颗粒可同时通过前述三种途径诱发铁死亡,显著提升铁死亡诱导剂针对肿瘤的疗效,因此,上述锰基纳米颗粒在制备抗肿瘤药物中的应用应在本发明的保护范围之内。
优选地,所述肿瘤包括肝癌、乳腺癌、胰腺癌或前列腺癌中的一种或几种。
最优选地,所述乳腺癌为三阴性乳腺癌(TNBC)。本发明将制得的锰基纳米颗粒用于治疗小鼠三阴性乳腺癌时,发现小鼠肿瘤明显消退,表现出优异的抗肿瘤效果。
本发明具有以下有益效果:
1.本发明所述锰基纳米颗粒MMMⅢM-SS NPs具有良好的水分散性、生物相容性、肿瘤选择性富集、氧化还原响应性,对谷胱甘肽和过氧化氢十分敏感,能通过三种途径(高效消耗肿瘤细胞内的GSH、H2O2,以及激活cGAS-STING通路)同时诱发铁死亡,进一步提升了药物疗效,为铁死亡纳米诊疗制剂提供了一种新的选择。
2.本发明所述锰基纳米颗粒对cGAS-STING通路的激活还能显着促进宿主抗原递呈细胞对肿瘤抗原的递呈能力,促进细胞毒性T细胞在肿瘤组织内的浸润和肿瘤特异性杀伤,放大免疫驱动的铁死亡。
3.本发明所述锰基纳米颗粒在肿瘤细胞中生成的MM2+具有磁共振成像探针的功能,可进行肿瘤成像,提高肿瘤部位T1磁共振成像效果,实现肿瘤精准可控的可视化治疗,成为诊疗一体化的纳米药物体系,为肿瘤的诊疗提供了一种新的选择。
4.本发明所述锰基纳米颗粒的制备方法简单,适于大量推广。
附图说明
图1为本发明锰基纳米颗粒诱发铁死亡的机制示意图。
图2为实施例1锰基纳米颗粒的制备流程图。
图3为实施例1锰基纳米颗粒的透射电镜图谱。
图4为实施例1锰基纳米颗粒的元素mappiMg图谱。
图5为实施例1锰基纳米颗粒的拉曼图谱。
图6a为MMMⅢM-SS NPs的X射线光电子能谱,图6b为MMMⅢM-SS NPs与MM相关的XPS谱图,图6c为MMMⅢM-SS NPs+GSH样品与MM相关的XPS谱图。
图7为实施例1锰基纳米颗粒的GSH消耗率统计图。
图8为实施例1锰基纳米颗粒的H2O2消耗率统计图。
图9A为实验A组的·OH含量变化情况,图9B为实验B组的·OH含量变化情况,图9C为实验C组的·OH含量变化情况。
图10A为实验A组的脂质过氧化物含量变化情况,图10B为实验B组的脂质过氧化物含量变化情况,图10C为实验C组的脂质过氧化物含量变化情况。
图11为T1加权核磁共振成像图。
图12为CD8+T细胞、IFNγ、SLC7A11的变化情况。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
激光共聚焦荧光显微镜购自德国蔡司LSM 510;
细胞计数器购自美国默克(Scepter 2.0);
小鼠乳腺癌细胞(4T1)购自中科院上海细胞库;
BALB/c小鼠购自广东省医学实验动物中心。
实施例1一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒的制备
S1.将MMMOAcM3·2H2O(3mmol)与3,3'-二硫代二丙酸(3mmol)溶解在15mLDMF中,超声40miM、在120℃下加热36h,将反应体系冷却至25℃,再在7000rpm下离心10miM,最后用甲醇和DMF反复洗涤至上清液无色以去除未反应的分子,得到MMMⅢM-SS;
S2.将甲氧基聚乙二醇(2.5mmol,分子量为5000)、MMMⅢM-SS(0.5mmol)、EDC(0.5mmol)、DMAP(0.5mmol)溶解在50mLTHF中,在避光环境中、45℃下边搅拌边加热36h,通过旋转蒸发器去除THF,转移至截留分子量(MWCO)为7000Da的透析袋中,用蒸馏水透析48h,且透析过程中,每8h更换一次蒸馏水,最后冷冻干燥,得到所述锰基纳米颗粒MMMⅢM-SSNPs。
制备流程如图2所示。
实施例2一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒的制备
S1.将MMMOAcM3·2H2O(6mmol)与3,3'-二硫代二丙酸(3mmol)溶解在15mLDMF中,超声30miM、在150℃下加热24h,将反应体系冷却至20℃,再在10000rpm下离心5miM,最后用甲醇和DMF反复洗涤至上清液无色以去除未反应的分子,得到MMMⅢM-SS;
S2.将甲氧基聚乙二醇(2.0mmol,分子量为1000)、MMMⅢM-SS(0.5mmol)、EDC(1mmol)、DMAP(1mmol)溶解在50mLTHF中,在避光环境中、40℃下边搅拌边加热48h,通过旋转蒸发器去除THF,转移至截留分子量(MWCO)为7000Da的透析袋中,用蒸馏水透析24h,且透析过程中,每4h更换一次蒸馏水,最后冷冻干燥,得到所述锰基纳米颗粒MMMⅢM-SS NPs。
实施例3一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒的制备
S1.将MMMOAcM3·2H2O(3mmol)与3,3'-二硫代二丙酸(3mmol)溶解在15mLDMF中,超声60miM、在110℃下加热48h,将反应体系冷却至30℃,再在6000rpm下离心20miM,最后用甲醇和DMF反复洗涤至上清液无色以去除未反应的分子,得到MMMⅢM-SS;
S2.将甲氧基聚乙二醇(4.0mmol,分子量为10000)、MMMⅢM-SS(0.5mmol)、EDC(0.5mmol)、DMAP(0.5mmol)溶解在50mLTHF中,在避光环境中、50℃下边搅拌边加热24h,通过旋转蒸发器去除THF,转移至截留分子量(MWCO)为7000Da的透析袋中,用蒸馏水透析72h,且透析过程中,每8h更换一次蒸馏水,最后冷冻干燥,得到所述锰基纳米颗粒MMMⅢM-SSNPs。
实验例1
取部分实施例1得到的MMMⅢM-SS NPs加水配制成1mg/mL的溶液,加入10mMGSH反应6h后,将反应体系转移至截留分子量(MWCO)为7000Da的透析袋中,用蒸馏水透析24h,且透析过程中,每8h更换一次蒸馏水,最后冷冻干燥,即得到MMMⅢM-SS NPs+GSH样品。
(1)采用透射电子显微镜(TEM)对实施例1所得MMMⅢM-SS NPs进行测试,得到的透射电镜图谱如图3所示,可见实施例1得到的MMMⅢM-SS NPs为类球形且分散单一。
(2)采用TEM对实施例1所得MMMⅢM-SS NPs进行测试,得到的元素mappiMg图谱如图4所示,可见实施例1得到的MMMⅢM-SS NPs中含有MM、S、O三种元素,且这三种元素在MMMⅢM-SS NPs中均匀分布。
(3)采用拉曼光谱仪对实施例1所得MMMⅢM-SS NPs进行测试,得到的拉曼图谱如图5所示,可见实施例1得到的MMMⅢM-SS NPs含有二硫键。
(4)采用X射线光电子能谱技术(XPS)对实施例1所得MMMⅢM-SS NPs和MMMⅢM-SSNPs+GSH样品进行测试,得到MMMⅢM-SS NPs的X射线光电子能谱如图6a所示,再通过分峰软件XPS Peak处理后的MMMⅢM-SS NPs与MM相关的XPS谱图如图6b所示,结合图6a和图6b可知实施例1得到的MMMⅢM-SS NPs中MM的价态为+3价;且图6c为分峰软件XPS Peak处理后的MMMⅢM-SS NPs+GSH样品与MM相关的XPS谱图,可见经GSH处理后MM的价态为+2价。
(5)根据说明书的方法,用GSH检测试剂盒对实施例1所得MMMⅢM-SS NPs和MMMⅢM-SS NPs+GSH样品进行测试,得到的GSH消耗率统计图如图7所示,可见实施例1得到的MMMⅢM-SS NPs可有效消耗GSH。
结合经GSH处理后MM的价态从+3价变为+2价,且GSH被有效消耗,可知MMMⅢM-SSNPs通过自身的金属离子(即锰离子)与肿瘤细胞内富集的GSH之间发生的氧化还原反应消化掉了GSH(即MM3++GSH→MM2++GSSH),从而抑制GPX4的活性,细胞抗过氧化能力降低,脂质ROS堆积,造成肿瘤细胞发生铁死亡。
(6)根据说明书的方法,用H2O2检测试剂盒对实施例1所得MMMⅢM-SS NPs和MMMⅢM-SS NPs+GSH样品进行测试,得到的H2O2消耗率统计图如图8所示,可见实施例1得到的MMMⅢM-SS NPs可有效消耗H2O2。
实验例2
一、实验方法
1、MMMⅢM-SS NPs溶液(1mg mL-1)的配制
取1mg实施例1所得MMMⅢM-SS NPs分散在1mL pH 7.4的去离子水中,用0.22μm的无菌过滤器过滤除菌,再将其分成多份,分别配制成0、50、100和200μg mL-1。
2、细胞培养
将小鼠乳腺癌细胞(4T1)置于含有10%胎牛血清、1%青霉素-链霉素双抗的RPMI-1640培养液中,在37℃、5%CO2的湿润环境中培养。实验前用细胞计数器测定细胞密度。
3、细胞内羟基自由基(·OH)、脂质过氧化物(LPO)含量的检测
将4T1细胞接种在激光共聚焦荧光显微镜(CLSM)的共聚焦培养皿上,培养12h,利用·OH检测探针(BBoxiProbe,BB-470563)测量4T1细胞内·OH含量的变化情况,且利用C11-BODIPY581/591检测探针(GlpBio TechMology)测量4T1细胞内脂质过氧化物含量的变化情况。分为3组:
(1)实验A组(不同浓度的MMMⅢM-SS NPs):分别在装有4T1细胞的培养皿中添加1mL的0、50、100和200μg mL-1的MMMⅢM-SS NPs溶液,培养12h;
(2)实验B组(不同培养时间):在装有4T1细胞的培养皿中添加1mL的50μg mL-1的MMMⅢM-SS NPs溶液,培养0、2、6和12h;
(3)实验C组(不同浓度的H2O2):分别在装有4T1细胞的培养皿中添加50μg mL-1的MMMⅢM-SS NPs溶液,再加入1mL的0、50、100和200mMH2O2溶液,培养12h。
培养结束后,将上述每组细胞分成两份,一份与·OH检测探针共孵育20miM,另一份与C11-BODIPY581/591检测探针共孵育20miM,两份细胞均用DAPI染色5miM后,用CLSM获得细胞的荧光图像,获取4T1细胞内·OH和脂质过氧化物含量的变化情况。
二、实验结果
实验A~C组4T1细胞内·OH含量的变化情况如图9所示,其中,图9A为实验A组的·OH含量变化情况,图9B为实验B组的·OH含量变化情况,图9C为实验C组的·OH含量变化情况。
实验A~C组4T1细胞内脂质过氧化物含量的变化情况如图10所示,其中,图10A为实验A组的脂质过氧化物含量变化情况,图10B为实验B组的脂质过氧化物含量变化情况,图10C为实验C组的脂质过氧化物含量变化情况。
由图9和10可知,不管是·OH的含量还是脂质过氧化物的含量,加入MMMⅢM-SSNPs的细胞均呈现出比对照组(MMMⅢM-SS NPs浓度、培养时间或H2O2浓度为0的细胞)更强的荧光强度,且随着MMMⅢM-SS NPs浓度、培养时间、H2O2浓度的增加,荧光强度(尤其是绿色荧光信号)越来越强,表明MMMⅢM-SS NPs在细胞内与H2O2发生了类芬顿反应(MM2++H2O2→MM3++·OH+OH-),形成具有细胞毒性的·OH,·OH的含量显著增加,·OH进而和细胞内脂质体反应生成脂质过氧化物,脂质过氧化物的含量也显著增加,从而引起细胞内脂质活性氧堆积,引发肿瘤细胞的铁死亡。
实验例3
一、实验方法
将4T1细胞(1.0×106细胞)用100μL磷酸盐缓冲盐溶液悬浮,皮下注射到BALB/c小鼠的侧翼内,建立动物肿瘤模型(三阴性乳腺癌),当肿瘤大小达到100mm3时,进行实验。
将实施例1所得MMMⅢM-SS NPs(10mg/kg)注入荷瘤小鼠的尾静脉,分别采用3.0TMRI临床扫描仪于注射前及注射后4h、8h、12h、24h对小鼠肿瘤部位进行磁共振成像(MRI)。
二、实验结果
得到的T1加权核磁共振成像图如图11所示,可见注射MMMⅢM-SS NPs的小鼠肿瘤部位MRI信号强度持续增强,在注射12h时达到最强,并在24h时仍保持较高的MRI信号水平,表明静脉尾注射MMMⅢM-SS NPs后,纳米药物可有效地在肿瘤部位积聚,利用MMMⅢM-SSNPs和肿瘤部位富集的GSH反应生成的MM2+能在磁共振成像中作为T1造影剂的特性,精准明确了肿瘤区域,可为后续的治疗提供有效指导,因此,MMMⅢM-SS NPs可作为一种肿瘤微环境敏感的T1造影剂,提高肿瘤部位T1磁共振成像效果,实现体内药物输送及治疗的无创监测,以及肿瘤精准可控的可视化治疗,成为诊疗一体化的纳米药物体系,为肿瘤的诊疗提供了一种新的选择。
实验例4
一、实验方法
将4T1细胞(1.0×106细胞)用100μL磷酸盐缓冲盐溶液悬浮,皮下注射到BALB/c小鼠的侧翼内,建立动物肿瘤模型(三阴性乳腺癌),当肿瘤大小达到100mm3时,将荷瘤小鼠随机分为3组,每组5只,进行实验。
三组小鼠分别尾静脉注射5mg/kg的PBS、MMCl2或实施例1所得MMMⅢM-SS NPs,每两天给药一次,直至第10天时将肿瘤组织取出、切片、置于二甲苯中脱蜡、梯度乙醇溶液(100%→80%)水化、切片,再用柠檬酸钠缓冲液(pH 6.0)进行酶抗原修复,25℃下在过氧化氢(5%)溶液浸泡15miM,然后将每组小鼠封闭后的肿瘤组织切片分别与抗CD8+、IFN-γ和SLC7A11抗体在4℃下孵育12h,接着用PBS漂洗3次后,用二抗aMti-CD8(FITCcoMjugated),aMti-IFN-γ(PE coMjugated),aMti-SLC7A11(Cy5 coMjugated))与肿瘤组织在25℃下孵育60miM,最后用DAPI进行核复染,在激光共聚焦荧光显微镜下观察并拍照。
2、实验结果
CD8+T细胞、IFNγ、SLC7A11的变化情况如图12所示,可见MMMⅢM-SS NPs处理组显著增强了CD8+T细胞在肿瘤部位的浸润,IFN-γ含量也有所增强,而SLC7A11的含量则显著降低,表明MMMⅢM-SS NPs在肿瘤细胞内生成的MM2+能有效激活细胞的cGAS-STING通路,活化CD8+T细胞,释放IFNγ下调SLC7A11的表达,从而抑制肿瘤细胞对胱氨酸的摄取,阻碍GSH的吸收,导致GPX4活性降低,细胞抗过氧化能力降低,脂质活性氧堆积,造成肿瘤细胞发生铁死亡。
此外,本发明还将实施例2和3所得MMMⅢM-SS NPs重复了实验例1~4的实验,得到的结果与实施例1相近似,表明MMMⅢM-SS NPs能通过三种途径(高效消耗肿瘤细胞内的GSH、H2O2,以及激活cGAS-STING通路)同时诱发铁死亡,进一步提升了药物疗效,为铁死亡纳米诊疗制剂提供了一种新的选择。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒,其特征在于,所述锰基纳米颗粒含有三价锰离子和二硫键。
2.一种谷胱甘肽和过氧化氢敏感的锰基纳米颗粒的制备方法,其特征在于,包括如下步骤:
S1.三价锰盐与二硫化物通过配位反应得到MMMⅢM-SS;
S2.甲氧基聚乙二醇与MMMⅢM-SS通过酯化反应得到所述锰基纳米颗粒。
3.根据权利要求2所述制备方法,其特征在于,所述三价锰盐包括三价醋酸锰、三价硫酸锰、三价磷酸锰中的一种或几种。
4.根据权利要求2所述制备方法,其特征在于,所述二硫化物包括3,3'-二硫代二丙酸、2,2'-二硫代二丙酸、3,3'-二硫代二丙酸、4,4'-二硫代二丁酸中的一种或几种。
5.根据权利要求2所述制备方法,其特征在于,所述三价锰盐与二硫化物的摩尔比为1~2:1。
6.根据权利要求2所述制备方法,其特征在于,S1为:三价锰盐与二硫化物在有机溶剂中超声、加热,并经后处理得到MMMⅢM-SS。
7.根据权利要求2所述制备方法,其特征在于,所述甲氧基聚乙二醇与MMMⅢM-SS的摩尔比为4~8:1。
8.根据权利要求2所述制备方法,其特征在于,S2为:甲氧基聚乙二醇、MMMⅢM-SS、活化剂、催化剂在有机溶剂中避光加热,并经后处理得到所述锰基纳米颗粒。
9.权利要求1所述锰基纳米颗粒在制备抗肿瘤药物中的应用。
10.根据权利要求9所述应用,其特征在于,所述肿瘤包括肝癌、乳腺癌、胰腺癌或前列腺癌中的一种或几种。
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