CN113750251B - 一种多功能纳米药物载体以及治疗药物的制备方法和应用 - Google Patents
一种多功能纳米药物载体以及治疗药物的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种多功能纳米药物载体及其治疗药物的制备方法和应用,该纳米载体制备包括:将GO与TCPP混合得到GO‑TCPP,再与Fe3+通过自主装得到GO‑MOF;将DSPE‑PEG‑FA加入到红细胞中得到DSPE‑PEG‑FA修饰的红细胞膜FA‑EM,向FA‑EM中加入GO‑MOF得到FA‑EM包裹的GO‑MOF。本发明制备的纳米载体能有效避免机体的免疫清除并实现药物靶向传递,将光能转化为热能,在肿瘤部位产生热量,将热能传递给周围的氧气,产生ROS诱导肿瘤死亡,促进肿瘤相关抗原释放并刺激抗原提呈细胞,诱导机体发生抗肿瘤免疫反应,该纳米载体不仅作为核磁成像造影剂,而且具有类过氧化氢酶的活性。
Description
技术领域
本发明属于生物纳米材料领域,具体涉及一种多功能纳米药物载体及其治疗药物的制备方法和应用。
背景技术
目前临床上治疗肿瘤的方式主要以手术切除为主,放疗,化疗等方法为辅,虽然在一定程度上能提高肿瘤患者的生存率,但是切除周边残留的肿瘤细胞会迅速复发并转移,给患者造成无尽的经济负担与身心痛苦。因此,开发新型治疗方案迫在眉睫,光线疗法因其具有高选择性,副作用小,且局部给药,被广泛应用于肿瘤的治疗,其基本原理是利用近红外光激发光敏材料产生热量,诱导肿瘤细胞发生凋亡。近年来,随着光热纳米材料及光敏剂的不断发展,为肿瘤的治疗提供了更理想的方法。因此,开发新型的多功能的近红外光诱导的纳米材料对肿瘤的治疗具有重要的意义。
金属有机骨架(metal-organic framework,MOF)因其具有较大的比表面积及较高的孔隙率和良好的稳定性,被广泛应用于药物的传递,且MOF可与不同的基质结合形成多功能复合物,因此在MOF的合成过程中可以加入一些治疗试剂,可以增加药物的装载量并具有一定的经济效益。卟啉作为典型的光敏剂,用于 MOF的合成,不仅使合成的MOF在近红外光的激发下产生大量的热,用于肿瘤的光热治疗,而且所制备的MOF具有游离卟啉的光学特性用于肿瘤的荧光成像。更重要的是,光线疗法产生的热可以直接诱导肿瘤发生凋亡,并在肿瘤部位释放肿瘤相关抗原,招募并激活抗原提呈细胞,诱导抗原特异性的抗肿瘤免疫反应。
尽管MOF作为抗肿瘤药物传递及治疗系统,但是当MOF进入机体血液循环后,能被机体免疫系统识别并清除,且缺乏靶向递送能力,严重限制了其应用及发展。
发明内容
发明目的:针对现有技术存在的问题,本发明提供一种多功能纳米药物载体的制备方法,本发明制备了一种多功能的光热药物载体,不仅能提高药物载体的靶向性及避免被机体免疫系统清除,而且根据特殊的TME增加肿瘤的诊疗效果。本发明设计的FA-EM@GO-MOF,在血液循环中能有效避免被机体的免疫系统识别并清除,有效的延长了其半衰期;到达肿瘤组织后能有效的与肿瘤细胞表面的叶酸受体结合,增强肿瘤细胞对FA-EM@GO-MOF的吞噬;FA-EM@GO-MOF 可与肿瘤组织中高浓度GSH反应,使MOF的荧光信号恢复,便于肿瘤的诊断;且FA-EM@GO-MOF具有类过氧化氢酶的活性,可与肿瘤组织高浓度H2O2反应,产生氧气,有效缓解肿瘤组织缺氧状态,增加肿瘤治疗效果。
本发明提供一种基于多功能纳米药物载体的肿瘤治疗药物。
本发明第三个目的提供制备的多功能纳米药物载体的应用。
技术方案:为了实现上述目的,本发明所述的一种多功能纳米载体 FA-EM@GO-MOF的制备方法,包括如下步骤:
(1)将GO与TCPP加入到磷酸盐缓冲液中,超声后,真空干燥,得到 GO-TCPP储存备用;
(2)将Fe(NO3)3·9H2O和C2H3NaO2·3H2O加入到甲醇溶液中,在回流反应;离心收集固体沉淀[Fe3O(CH3COO)6(H2O)3](CH3COO),并用清洗,真空干燥,备用;
(3)将[Fe3O(CH3COO)6(H2O)3](CH3COO)与GO-TCPP加入到DMF溶液中,并加入甲酸,充分混匀,将此混合溶液高温反应,得到GO-MOF纳米载体;
(4)将红细胞与超纯水混合,静置后超纯水清洗,至上清无色透明;
(5)将DSPE-PEG-FA加入到步骤(5)的红细胞溶液中并搅拌,清洗,得到DSPE-PEG-FA修饰的红细胞膜FA-EM;
(6)将FA-EM重悬在缓冲液中,并向上述溶液加入步骤(3)GO-MOF纳米载体搅拌,得到FA-EM包裹的GO-MOF(FA-EM@GO-MOF)。
其中,步骤(1)中GO与TCPP的质量比为1:0.5-1.5。
作为优选,步骤(1)中GO与TCPP的质量比为1:1。
其中,步骤(2)中Fe(NO3)3·9H2O与C2H3NaO2·3H2O的质量比为1-2:1
作为优选,步骤(2)中Fe(NO3)3·9H2O与C2H3NaO2·3H2O的质量比为1.5: 1。
其中,步骤(3)中[Fe3O(CH3COO)6(H2O)3](CH3COO)与GO-TCPP的质量比为2-3:1。
作为优选,步骤(3)中[Fe3O(CH3COO)6(H2O)3](CH3COO)与GO-TCPP的质量比为2.5:1。
其中,步骤(3)中将混合溶液高温反应放入反应釜中,在80-100℃的烘箱中反应20-24h,得到GO-MOF纳米载体。
本发明所述的制备方法所制备的多功能纳米载体FA-EM@GO-MOF。
本发明所述的多功能纳米载体FA-EM@GO-MOF在制备核磁成像的造影剂、环境响应型载药试剂和光热治疗、光动力治疗试剂中的应用。
本发明所述的用于肿瘤的治疗药物,所述治疗药物以制备的多功能纳米载体 FA-EM@GO-MOF为载体并装载抗肿瘤药物。
其中,所述抗肿瘤药物为DOX。
本发明所述的治疗药物的制备方法,包括如下步骤:
(1)将GO与TCPP加入到磷酸盐缓冲液中,超声后,真空干燥,得到 GO-TCPP储存备用;
(2)将Fe(NO3)3·9H2O和C2H3NaO2·3H2O加入到甲醇溶液中,在回流反应;离心收集固体沉淀[Fe3O(CH3COO)6(H2O)3](CH3COO),并清洗,真空干燥,备用;
(3)将[Fe3O(CH3COO)6(H2O)3](CH3COO)与GO-TCPP加入到DMF溶液中,并加入甲酸,充分混匀,将此混合溶液高温反应,得到GO-MOF纳米载体;
(4)GO-MOF溶解于磷酸盐缓冲液中充分混匀,并加入DOX,避光搅拌得到GO-MOF/DOX溶液;
(5)将GO-MOF/DOX溶液,离心收集沉淀,清洗,直至上清无色透明;
(6)将红细胞与超纯水混合,静置后,用超纯水清洗,至上清无色透明;
(7)将DSPE-PEG-FA加入到步骤(6)的红细胞溶液中并搅拌,清洗,得到DSPE-PEG-FA修饰的红细胞膜FA-EM;
(8)将FA-EM重悬在缓冲液中,并向上述溶液加入GO-MOF/DOX搅拌,得到FA-EM包裹的GO-MOF(FA-EM@GO-MOF/DOX)。
本发明制备的药物载体是一种多功能环境响应型诊疗药物载体,其以 GO-TCPP和Fe3+通过自主装形成荧光诊疗试剂。利用GO的荧光淬灭性能,合成GO-MOF纳米载体,继而用FA-EM进一步修饰GO-MOF,使合成的 FA-EM@GO-MOF药物载体不仅具有免疫逃逸的功能,而且能靶向传递到肿瘤部位,与肿瘤细胞表面的叶酸受体结合,增加肿瘤细胞对FA-EM@GO-MOF纳米载体的吞噬,同时由于载体经过叶酸修饰的红细胞膜(FA-EM)包裹后, FA-EM@GO-MOF进入血液循环,避免被单核吞噬系统清除,并且能在肿瘤部位聚集并被肿瘤细胞吞噬。当到达肿瘤组织或被肿瘤细胞吞噬后,高浓度GSH 使GO还原成rGO使MOF从GO表面解离,荧光信号恢复,通过共聚焦成像及荧光分光光度仪器检测荧光恢复情况,用于肿瘤的荧光成像。FA-EM@GO-MOF 纳米载体中的Fe3+不仅能与肿瘤微环境中高浓度H2O2反应,生成氧气,能有效缓解肿瘤组织缺氧状态,当被近红外光照射,产生大量的热量及ROS,杀伤肿瘤细胞,而且具有核磁成像的功能,能实时检测纳米材料在体内的运输过程,如能实时检测FA-EM@GO-MOF装载了阿霉素的FA-EM@GO-MOF/DOX在体内的传输,分布及代谢过程。因此,FA-EM@GO-MOF药物载体不仅可以装载药物,而且具有双模成像指导化疗,光热治疗和光动力联合治疗。同时本发明制备的治疗药物或者试剂FA-EM@GO-MOF/DOX具有良好的光热转化效率,具有优越的肿瘤联合治疗效果。
本发明制备的多功能FA-EM@GO-MOF药物载体,其中基于氧化石墨烯(GO)为基础形成多功能金属有机框架(GO-MOF)。GO-MOF不仅有较大的比表面积和荧光淬灭的能力,而且经过叶酸修饰的红细胞膜(FA-EM)包裹后形成的FA-EM@GO-MOF纳米载体,能有效的避免机体的免疫清除并实现药物的靶向传递。FA-EM@GO-MOF纳米载体装载的抗肿瘤药物(DOX)到达肿瘤部位后,在近红外光激发下(NIR),可实现化疗(CT),光热治疗(PTT)和光动力治疗(PDT)三种方法联合治疗肿瘤。由于肿瘤微环境(TME)含有大量的谷胱甘肽(GSH),过氧化氢(H2O2),缺氧和偏酸(pH 5.5)等特点,容易对肿瘤治疗产生耐药。因此,当FA-EM@GO-MOF/DOX纳米载体到达小鼠肿瘤部位后,根据特殊的TME使FA-EM@GO-MOF/DOX纳米载体逐渐降解,释放出装载的DOX用于CT,高浓度的GSH使MOF从FA-EM@GO-MOF/DOX表面解离,荧光信号恢复用于肿瘤的诊疗,当肿瘤部位暴露于NIR下,MOF作为光敏剂将光能转化为热能,在肿瘤部位产生大量的热量,并将热能传递给周围的氧气,产生活性氧(ROS)诱导肿瘤细胞死亡,并使肿瘤相关抗原释放刺激抗原提呈细胞,诱导机体发生抗肿瘤免疫反应。同时,FA-EM@GO-MOF纳米载体不仅作为核磁成像的造影剂,实时监测肿瘤治疗,而且具有类过氧化氢酶的活性,可与肿瘤微环境中高浓度H2O2反应,生成氧气,缓解肿瘤微环境缺氧状态的同时,增加光动力治疗的结果,具体是与TME中H2O2反应生成氧气,缓解肿瘤组织缺氧状态,增加PDT治疗效果。
具体地,本发明中FA-EM@GO-MOF/DOX用于肿瘤的治疗,当其进入血液循环后,避免被机体的免疫系统清除并能在肿瘤部位聚集,实时监测药物在体内的运输及在肿瘤组织内的释放与降解。因此,根据特殊的TME FA-EM@GO-MOF/DOX释放出装载的抗肿瘤药物,并缓解因缺氧和高浓度GSH 导致的耐药,其中H2O2聚集在红细胞膜表面,使红细胞膜孔径变大,有利于药物渗透到肿瘤组织;GSH使Fe3+还原成,导致FA-EM@GO-MOF/DOX结构坍塌, DOX释放;酸性环境使DOX发生质子化作用,DOX进一步从 FA-EM@GO-MOF/DOX上释放,根据特殊的TME,FA-EM@GO-MOF/DOX释放出装载的抗肿瘤药物,并与肿瘤组织高浓度H2O2反应,产生氧气,有效缓解肿瘤组织缺氧状态。当FA-EM@GO-MOF/DOX暴露于近红外激发光下,可产生热量和ROS诱导肿瘤细胞凋亡,并释放肿瘤相关抗原,诱导机体抗肿瘤免疫反应。同时在血液运输过程中,FA-EM@GO-MOF/DOX的荧光信号处于淬灭的状态,减少运输过程中光毒性。当进入肿瘤组织或肿瘤细胞时,根据肿瘤微环境中高浓度GSH,药物载体的荧光信号恢复,用于肿瘤的荧光成像及光热和光动力治疗。
本发明中使用的TCPP作为典型的光敏剂,其与金属离子通过自主装形成的 MOF,不仅具有较高的比表面积,而且具有荧光成像及良好的光热转化效率,可用于肿瘤的光热治疗。但是其在运输过程中,易引起光毒性,GO具有荧光淬灭的功能,其与TCPP组合,并与Fe3+通过自主装的方式形成的GO-MOF具有荧光淬灭的功能,有效降低运输过程中的光毒性。同时肿瘤组织中,高浓度GSH 作为控制荧光恢复的开关,使GO还原成还原型石墨烯(rGO),MOF从GO表面解离,荧光信号恢复,有利于肿瘤的诊断。
本发明中的GO-MOF经过FA-EM修饰后,能有效避免被机体免疫系统清除,有效聚集在肿瘤组织,与肿瘤细胞特异性结合;肿瘤组织高浓度GSH作为荧光控制的开关,使MOF荧光信号恢复,有利于肿瘤的诊断;FA-EM@GO-MOF具有类酶的性质,能与肿瘤组织高浓度H2O2反应,产生氧气,有效缓解肿瘤组织缺氧情况;FA-EM@GO-MOF不仅可作为纳米载体有利于药物运输,而且作为级联反应器,有效提高肿瘤治疗效果。
原理:本发明中1)GO的荧光淬灭功能,2)FA-EM@GO-MOF纳米载体较大比表面积适用于药物装载,3)叶酸修饰的红细胞膜包裹,具有免疫逃逸及肿瘤靶向作用,4)GSH控制的荧光信号恢复,有利于肿瘤诊断;5)FA-EM@GO-MOF 纳米载体具有类过氧化氢酶性质,有效缓解肿瘤组织缺氧状态。GO具有荧光淬灭作用,能使合成的GO-MOF具有荧光淬灭的效果;叶酸修饰的红细胞膜包裹 GO-MOF,具有免疫逃逸及靶向传递功能;肿瘤组织高浓度GSH作为还原剂,能使MOF从GO表面解离,使MOF的荧光信号恢复,有利于肿瘤的诊断; FA-EM@GO-MOF纳米载体具有类过氧化氢酶活性,可与肿瘤组织H2O2反应,生成氧气,有效缓解肿瘤组织缺氧状态,为后期PDT治疗提供条件。本发明制备的FA-EM@GO-MOF纳米载体具有免疫逃逸及靶向传递功能;可通过GSH 控制荧光信号恢复功能,可用于肿瘤的诊断;FA-EM@GO-MOF纳米载体可与 H2O2反应,生成氧气,缓解肿瘤组织缺氧状态,近红外光照射下产生大量ROS,有效抑制肿瘤生长。
有益效果:与现有技术相比,本发明具有如下优点:
(1)FA-EM@GO-MOF纳米载体能有效避免被机体的免疫系统清除,实现免疫逃逸延长血液循环时间,并具有靶向性能在肿瘤部位聚集,与肿瘤细胞表面的叶酸受体结合,增加肿瘤细胞对载体的吞噬。
(2)FA-EM@GO-MOF纳米载体具有较大的比表面积,不仅能够有效的增加抗肿瘤药物的负载能力,而且具有较强的MRI和荧光成像功能。
(3)FA-EM@GO-MOF纳米载体具有类似过氧化氢酶的活性,可以肿瘤微环境中高浓度H2O2反应,生成大量的O2,有效克服缺氧环境,能有效增强PDT 治疗效果。
(4)FA-EM@GO-MOF纳米载体具有较高的比表面积,对抗肿瘤药物DOX 具有较高的药物装载量。
(5)FA-EM@GO-MOF/DOX浸入到体外模拟肿瘤微环境中后,能根据肿瘤微环境中偏酸,和高GSH浓度,释放出DOX,有效降低DOX对正常组织的损伤。
(6)当FA-EM@GO-MOF/DOX到达肿瘤部位后,在近红外光的激发下,能同时触发PDT和PTT协同治疗,促进肿瘤相关抗原的产生,在原位呈现免疫疫苗样功能。
(7)FA-EM@GO-MOF/DOX能促进DOX在肿瘤组织的渗透,有效弥补近红外光的渗透缺陷,有效抑制小鼠移植瘤的生长,并对机体的毒副作用较小。
(8)本发明的纳米载体和含药载体制备简单,使用方便,可以大量工业化生产。
附图说明
图1为本发明FA-EM@GO-MOF纳米载体比表面积示意图;
图2为本发明FA-EM@GO-MOF纳米载体荧光淬灭性能检测图;
图3为本发明FA-EM@GO-MOF纳米载体GSH响应图;
图4为本发明FA-EM@GO-MOF纳米载体核磁成像示意图;
图5为本发明FA-EM@GO-MOF纳米载体产氧能力示意图;
图6为本发明FA-EM@GO-MOF纳米载体光热转换效果图;
图7为本发明FA-EM@GO-MOF纳米载体环境控制药物释放功能;
图8为本发明FA-EM@GO-MOF纳米载体协同治疗效果示意图;
图9为本发明FA-EM@GO-MOF纳米载体在近红外作用下诱导树突状细胞成熟示意图;
图10为本发明FA-EM@GO-MOF纳米载体具有免疫逃逸及与肿瘤细胞特异性结合示意图。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂家建议的条件。
本发明中技术术语的缩写如下:金属有机骨架:MOF;肿瘤微环境:TME;氧化石墨烯:GO;还原型石墨烯:rGO;卟啉:TCPP;红细胞膜:EM;谷胱甘肽:GSH;DSPE-PEG-FA:FA;过氧化氢:H2O2。
实施例中氧化石墨烯:GO直接市售购买或者按现有文献如Moon,I.K.,et al.,Reduced graphene oxide by chemical graphitization.Nat Commun,2010.1:p.73制备均可。
卟啉TCPP购于阿拉丁(Dye content 75%,CAS:14609-54-2)
DSPE-PEG-FA:1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000]阿拉丁(1236288-25-7)。
BALB/c小鼠采用6周龄雄性。
实施例1
(1)将10mg的GO与10mg的TCPP加入到10mL的PBS溶液(pH7.4)中,320W超声3h,真空干燥得到GO-TCPP,储存备用,
(2)将100mg的Fe(NO3)3·9H2O和400mg的C2H3NaO2·3H2O加入到30mL 的甲醇溶液中,在100℃下回流12h;离心收集固体沉淀得到 [Fe3O(CH3COO)6(H2O)3](CH3COO),并用甲醇清洗三遍,真空干燥,备用;
(3)将100mg的[Fe3O(CH3COO)6(H2O)3](CH3COO)与40mg的GO-TCPP 加入到20mL的DMF溶液中,并加入2mL的甲酸,充分混匀,将此混合溶液放入反应釜中,在80℃的烘箱中反应24h,得到GO-MOF纳米载体;
(4)收集BALB/c小鼠的新鲜血液,用pH7.4的PBS清洗三遍,离心收集红细胞(约5×1012个);
(5)收集的红细胞与等质量超纯水混合,于4℃静置1h,释放红细胞内血红蛋白等相关成分,并用超纯水清洗,至上清无色透明,并5000rpm离心5min 收集下层红细胞,将得到的红细胞溶解在5mLpH7.4的PBS缓冲溶液中,得到红细胞溶液;
(6)将10mg的DSPE-PEG-FA加入到2.5mL的红细胞溶液中在500rpm下搅拌12h,用pH7.4的PBS清洗3遍,得到白色沉淀状的DSPE-PEG-FA修饰的红细胞膜(FA-EM);
(7)将10mg的FA-EM重悬在1mL的pH7.4的PBS溶液中,并向上述溶液加入1mg的GO-MOF纳米载体,在500rpm的转速下搅拌24h,离心后收集固体沉淀得到FA-EM包裹的GO-MOF(FA-EM@GO-MOF)。
本实施例1制备的FA-EM@GO-MOF的比表面积为1044cm2/g(图1),由于其较大的比表面积适用于药物的装载。
实施例2
(1)将100mg的Fe(NO3)3·9H2O和400mg的C2H3NaO2·3H2O加入到30 mL的甲醇溶液中,在100℃下回流12h;离心收集固体沉淀得到 [Fe3O(CH3COO)6(H2O)3](CH3COO),并用甲醇清洗三遍,真空干燥,备用;
(2)将100mg的[Fe3O(CH3COO)6(H2O)3](CH3COO)与40mg的TCPP加入到20mL的DMF溶液中,并加入2mL的甲酸,充分混匀,将此混合溶液放入反应釜中,在80℃的烘箱中反应24h,得到MOF纳米载体;
(4)收集BALB/c小鼠的新鲜血液,用pH7.4的PBS清洗三遍,离心收集红细胞(约5×1012个);
(5)收集的红细胞与等质量超纯水混合,于4℃静置1h,释放红细胞内血红蛋白等相关成分,并用超纯水清洗,至上清无色透明,并5000rpm离心5min 收集下层红细胞,将得到的红细胞溶解在5mLpH7.4的PBS缓冲溶液中,得到红细胞溶液;
(5)将10mg的DSPE-PEG-FA加入到2.5mL的红细胞溶液中在500rpm 下搅拌12h,用pH7.4的PBS清洗3遍,得到白色沉淀状的DSPE-PEG-FA修饰的红细胞膜(FA-EM);
(6)将10mg的FA-EM重悬在1mL的pH7.4的PBS溶液中,并向上述溶液加入1mg的MOF纳米载体,在500rpm的转速下搅拌24h,离心后收集固体沉淀得到具有荧光信号的FA-EM包裹的MOF(FA-EM@MOF)。
将TCPP、实施例1制备的FA-EM@GO-MOF和实施例2制备的 FA-EM@MOF通过荧光光谱仪检测,所制备的FA-EM@GO-MOF具有荧光淬灭的功能,而FA-EM@MOF的荧光信号能被检测到,说明FA-EM@GO-MOF具有荧光淬灭的功能(图2)。因此本发明后期用FA-EM@MOF、FA-EM@GO-MOF 分别与巨噬细胞和肿瘤细胞共孵育,通过检测荧光信号的恢复,来评估其免疫逃逸及靶向吞噬功能(详见实施例11)。
实施例3
(1)将10mg的GO与10mg的TCPP加入到10mL的pH7.4的PBS溶液中,320W超声3h,真空干燥得到GO-TCPP,储存备用,
(2)将100mg的Fe(NO3)3·9H2O和400mg的C2H3NaO2·3H2O加入到30mL 的甲醇溶液中,在100℃下回流12h;离心收集固体沉淀得到 [Fe3O(CH3COO)6(H2O)3](CH3COO),并用甲醇清洗三遍,真空干燥,备用;
(3)将100mg的[Fe3O(CH3COO)6(H2O)3](CH3COO)与40mg的GO-TCPP 加入到20mL的DMF溶液中,并加入2mL的甲酸,充分混匀,将此混合溶液放入反应釜中,在80℃的烘箱中反应24h,得到GO-MOF纳米载体;
(4)将40mg GO-MOF纳米载体分散于40mL pH7.4的PBS缓冲液中充分混匀,并加入20mg的DOX,避光在500rpm的转速下搅拌24h得到 GO-MOF/DOX溶液;
(5)将GO-MOF/DOX溶液,10000rpm离心10min,收集沉淀,用pH7.4 的PBS清洗数次,直至上清无色透明,弃去上清夜,收集下层的沉淀;
(6)收集BALB/c小鼠的新鲜血液,用pH7.4的PBS清洗三遍,离心收集红细胞(约5×1012个);
(7)收集的红细胞与等质量超纯水混合,于4℃静置1h,释放红细胞内血红蛋白等相关成分,并用超纯水清洗,至上清无色透明,并5000rpm离心5min 收集下层红细胞,将得到的红细胞溶解在5mLpH7.4的PBS缓冲溶液中,得到红细胞溶液;
(8)将10mg的DSPE-PEG-FA加入到5mL的红细胞溶液中,在500rpm 的转速下搅拌12h,用pH7.4的PBS清洗3遍,得到白色沉淀状的DSPE-PEG-FA 修饰的红细胞膜(FA-EM);
(9)将10mg FA-EM重悬在1mLpH7.4 PBS溶液中,并向上述溶液加入 1mg步骤(5)的GO-MOF/DOX在500rpm的转速下搅拌24h,离心后收集固体沉淀得到FA-EM包裹的GO-MOF(FA-EM@GO-MOF/DOX)。
实施例4
实施例4与实施例1的制备方法相同,不同之处在于:步骤(1)中GO与 TCPP的质量比为1:0.5。步骤(2)中Fe(NO3)3·9H2O与C2H3NaO2·3H2O的质量比为1:1。步骤(3)中[Fe3O(CH3COO)6(H2O)3](CH3COO)与GO-TCPP的质量比为2:1。
实施例5
实施例5与实施例1的制备方法相同,不同之处在于:步骤(1)中GO与 TCPP的质量比为1:1.5。步骤(2)中Fe(NO3)3·9H2O与C2H3NaO2·3H2O的质量比为2:1。步骤(3)中[Fe3O(CH3COO)6(H2O)3](CH3COO)与GO-TCPP的质量比为3:1;步骤(3)中将混合溶液高温反应放入反应釜中,在100℃的烘箱中反应20h,得到GO-MOF纳米载体。
实施例6
GSH介导的荧光恢复策略,体外检测按如下方法进行:
(1)将1mL的FA-EM@GO-MOF溶液(1mg/mL,实施例1制备的 FA-EM@GO-MOF,溶剂为pH7.4的PBS溶液)加入到4mL含有不同浓度GSH (0,2,4,6,8,10,12,14,16和18mM)的pH7.4的PBS缓冲溶液中,置于37℃的水平摇床上;
(2)8小时后,从上述各个样品管中,吸取200μL的含FA-EM@GO-MOF 纳米载体的样品,加入到2800μL的乙醇溶液中,通过荧光分析仪检测荧光信号随着不同GSH量的变化,FA-EM@GO-MOF纳米载体荧光信号的强弱。如图3所示,随着GSH含量的逐渐增加,FA-EM@GO-MOF的荧光信号逐渐增强,此结果说明,肿瘤组织中高浓度的GSH能作为FA-EM@GO-MOF荧光信号的开光,肿瘤细胞高浓度GSH使载体的荧光信号恢复,而正常组织中GSH的含量低,不足以控制FA-EM@GO-MOF/DOX荧光信号的恢复,有利于肿瘤的诊断。
实施例7
FA-EM@GO-MOF纳米载体体内核磁共振成像,具体步骤如下:
(1)取4T1移植瘤Balb/c小鼠(实验前,禁食24h),1mL的注射器吸取200μL的水合氯醛溶液(10%),通过腹腔注射到4T1移植瘤小鼠体内;
(2)待小鼠麻醉后,将其呈仰卧位平躺于小鼠固定板上并固定好小鼠四肢,将固定好的小鼠置于3.0T核磁共振仪器用于拍照的线圈中央附近;
(3)扫描完成后,将200μL的FA-EM@GO-MOF(1mg/mL,实施例1制备的FA-EM@GO-MOF,溶剂为pH7.4的PBS溶液)溶液,通过小鼠尾静脉注射到4T1移植瘤小鼠体内。分别于注射0h,12h,24h,36h和48h后进行核磁扫描,得到肿瘤部位相关图像,结果如图4所示。
以上结果说明,在未注射FA-EM@GO-MOF纳米载体时,在肿瘤部位可以检测到很强的的信号,随着FA-EM@GO-MOF纳米载体的注入,肿瘤部位的信号强度逐渐变暗,在注射后24h,肿瘤组织信号最暗,然后逐渐恢复。此结果说明,FA-EM@GO-MOF纳米载体具有良好的肿瘤靶向效果,在注射到肿瘤组织 24h后,能在肿瘤部位大量聚集,随后逐渐代谢,肿瘤组织信号逐渐恢复。
实施例8
FA-EM@GO-MOF纳米载体产氧能力,具体步骤如下:
(1)将实施例1制备的10mg FA-EM@GO-MOF纳米载体分别加入到30mL pH=7.4,pH=5.5的PBS溶液中(不含H2O2),以及30mL pH=7.4,pH=5.5 的PBS溶液中(含有30μMH2O2),以PBS(pH7.4)和PBS(pH7.4)含30μ M H2O2为空白对照。
(2)将溶解氧检测仪插入到溶液(1)中,在设计好的时间点(0-21min),检测溶液中的含氧量。
结果如图5所示,当溶液中不含有30μM H2O2时,溶液中的氧浓度保持不变,然而将FA-EM@GO-MOF纳米载体加入到含有30μM H2O2的溶液中后,可在溶液中产生氧气,此结果说明,FA-EM@GO-MOF纳米载体具有类过氧化氢酶的活性,可与溶液中H2O2反应生成氧气。
实施例9
FA-EM@GO-MOF纳米载体光热转换效率
检测FA-EM@GO-MOF纳米载体光热转换效率的方法步骤如下:
(1)将1mg的实施例1制备的GO-MOF纳米载体溶解于1mL的pH7.4的 PBS缓冲溶液中,充分混匀,
(2)吸取100μl的GO-MOF混合溶液,加入900μl的PBS缓冲液中,
(3)将配置好的100μg/mL的GO-MOF混合溶液置于红外激发器下,将功率密度调节为1.5W/cm2,
(4)每隔2min用红外热成像仪器记录温度变化,并拍照。
(5)FA-EM@GO-MOF的光热转化效率参考GO-MOF的操作步骤。
其结果如图6所示,当GO-MOF纳米载体经过1.5W/cm2的近红外光照射 10min后温度上升27.8℃,而GO-MOF经过FA-EM包裹后,在相同条件下, FA-EM@GO-MOF纳米载体温度上升26℃,此结果说明GO-MOF纳米载体经过 FA-EM包裹后,其光热转化效率并没有受到影响。而经过1.0W/cm2的近红外光照射10min后,温度仅身高10.7℃,说明FA-EM@GO-MOF纳米载体的光热转化效率受到近红外光强度及时间的影响,红外光强度越强及时间越长,光热转化效率越好。
实施例10
不同环境下DOX的释放
(1)称取10mg实施例3制备FA-EM@GO-MOF/DOX,分别溶解于30mL 不同溶液中,(a)PBS溶液(pH=7.4),(b)PBS溶液(pH=7.4)含高浓度 GSH(10mM),(c)PBS溶液(pH=7.4)含H2O2(30μM)溶液,(d)PBS 溶液(pH=5.5)及(d)PBS溶液pH=5.5,含GSH和H2O2(10mM GSH和30 μM H2O2)的混合溶液中;(2)在设计好的时间点,分别吸取上述混合溶液,用紫外-可见分光光度仪检测490nm处DOX的吸光值,分析DOX的释放。
其结果如图7所示,当FA-EM@GO-MOF/DOX,浸入到pH=7.4的,仅有 11%的DOX释放。而FA-EM@GO-MOF/DOX浸入到pH=5.5的环境中后,约有56%的DOX释放,主要是DOX在酸性环境中发生质子化,导致DOX释放。当FA-EM@GO-MOF/DOX浸入到H2O2溶液中,由于H2O2聚集在FA-EM表面导致DOX释放。当FA-EM@GO-MOF/DOX浸入GSH溶液中,引起 FA-EM@GO-MOF/DOX结构发生了坍塌,引起DOX释放。而 FA-EM@GO-MOF/DOX浸入到GSH,H2O2和酸性溶液的混合溶液中后中,约有67%的DOX释放。因此,FA-EM@GO-MOF可作为理想的药物载体,具有环境控制药物释放的能力。
实施例11
体内FA-EM@GO-MOF/DOX化疗-光疗联合抗肿瘤治疗
(1)4T1细胞用胰酶消化,用pH7.4的磷酸盐缓冲液重悬,并均匀混合;
(2)取出Balb/c小鼠,用75%酒精擦拭小鼠乳腺处,吸取200μL的4T1 (细胞数1×107)细胞悬液,注射到乳腺皮肤下;
(3)每天观察Balb/c小鼠移植瘤的生长情况,并用游标卡尺测量肿瘤组织的长边和短边直径(mm)并计算出小鼠肿瘤体积大小(mm3),小鼠移植瘤体积计算公式如下:V=0.5×a×b2。V:肿瘤体积,a:肿瘤长边直径,b:肿瘤短边直径;
(4)按照上述步骤构建小鼠移植瘤模型,小鼠移植瘤体积大小大约为~50mm3时,将移植瘤小鼠随机分为7组,分别为PBS(pH7.4)组,PBS+Laser (1.5W/cm2,10min)组,DOX(1mg/kg)组,FA-EM@GO-MOF/DOX(4mg/kg,实施例3制备)组,GO-MOF/DOX+Laser(4mg/kg,1.5W/cm2,10min,实施例 3制备)组,FA-EM@GO-MOF+Laser(4mg/kg,1.5W/cm2,10min,实施例3 制备)组和FA-EM@GO-MOF/DOX+Laser(4mg/kg,1.5W/cm2,10min,实施例3制备)组(808nm近红外激发光照射,简称:Laser),每组小鼠为6只;
(2)将上述各处理组的药物或者纳米载体采用pH7.4的PBS缓冲液配制成溶液,通过上述剂量尾静脉注射到小鼠体内,每3天注射一次,共注射7次,808 nm近红外激发光照射,在纳米载体注射小鼠体内24h后操作;
(3)每3天称取每组治疗组中每只小鼠的体重和肿瘤大小的变化,并做好相应的标记;
(4)22天后,脱颈处死小鼠,切除各治疗组中的肿瘤组织,并对各治疗组中,每只小鼠的肿瘤组织称重。
其结果如图8A所示,4T1小鼠移植瘤模型每三天经过尾静脉注射不同的药物进行治疗,每三天治疗一次,共治疗7次,22天后处死4T1小鼠移植瘤小鼠,并称取肿瘤体重及大小。如图8B-D所示,通过观察小鼠移植瘤的生长情况及重量大小,发现FA-EM@GO-MOF/DOX+Laser联合治疗后,能有效抑制小鼠移植瘤的生长;如图8E所示,虽然DOX治疗也能有效抑制肿瘤的生长,但是小鼠的体重急剧下降,而FA-EM@GO-MOF/DOX+Laser联合治疗组小鼠的体重没有明显的改变。此结果说明FA-EM@GO-MOF/DOX+Laser联合治疗能有效的抑制小鼠移植瘤生长,而且能有效的降低对机体的毒副作用。
实施例12
4T1移植瘤小鼠中腹股沟淋巴结中成熟DCs的百分率
(1)构建4T1小鼠移植瘤模型(同实施例11),小鼠移植瘤体积大小大约为~50mm3时,将移植瘤小鼠随机分为7组,分别经过PBS,PBS+Laser(1.5 W/cm2,10min),DOX(1mg/kg),FA-EM@GO-MOF/DOX(4mg/kg), GO-MOF/DOX+Laser(4mg/kg,1.5W/cm2,10min),FA-EM@GO-MOF+Laser (4mg/kg,1.5W/cm2,10min)和FA-EM@GO-MOF/DOX+Laser(4mg/kg,1.5 W/cm2,10min)治疗(808nm近红外激发光照射,简称:Laser),将上述各处理组的药物或者纳米载体采用pH7.4的PBS缓冲液配制成溶液,通过上述剂量尾静脉注射到小鼠体内,3天后,处死小鼠,收集腹股沟淋巴结;
(2)将不同处理组的小鼠腹股沟淋巴结置于无菌研磨管中研磨成单细胞悬液,将单细胞悬液通过200目的过滤器,离心收集细胞(1000rpm/10min),管底可见红色沉淀,轻轻弹均匀;
(3)吸取红细胞裂解液与细胞悬液(3:1)混合静置2min,加入10mL PBS 终止反应,离心(1000rpm/5min)去除红细胞;
(4)控制细胞密度并按照每管1×106细胞进行相应的流逝抗体 APC-anti-CD11c,PE-anti-CD80和FITC-anti-CD86染色,并用流逝细胞计数仪分析成熟DCs的比列。
其结果如图9所示,FA-EM@GO-MOF/DOX+Laser联合治疗后,诱导肿瘤细胞死亡并释放肿瘤相关抗原,刺激抗原提呈细胞,主要是树突状细胞(DCs)成熟,因此,分析各组小鼠腹股沟淋巴结肿DCs的表达量,从图中可以看出, FA-EM@GO-MOF/DOX+Laser联合治疗后,成熟DCs的表达量明显升高,为 13.1%,是PBS组的两倍左右。因此,本发明的FA-EM@GO-MOF/DOX通过化疗-光热-光动力联合治疗,能有效诱导DCs成熟,为后期抗肿瘤免疫提供基础。
实施例13
由于FA-EM@GO-MOF具有荧光淬灭的效果,采用具有荧光信号的 FA-EM@MOF(实施例2制备)代替FA-EM@GO-MOF(实施例1制备)来进行免疫逃逸及靶向吞噬作用,其中MOF(实施例1步骤2制备)和FA-EM@MOF 纳米载体采用pH7.4的PBS缓冲液配制成溶液。
免疫逃逸
(1)按终浓度100μg/mL分别将MOF和FA-EM@MOF纳米载体加入到含有1×105个巨噬细胞的细胞培养皿中;
(2)在37℃含有5%二氧化碳的细胞培养箱中孵育3h,弃去细胞培养基,并用7.4的PBS缓冲液清洗3遍,置于共聚焦激光扫描显微镜(CLSM)分别观察每个细胞培养皿的荧光信号。
靶向摄取
(1)按终浓度100μg/mL将FA-EM@MOF纳米载体分别加入到含有1×105个GES-1细胞和1×105个4T1细胞的培养皿中;
(2)在37℃含有5%二氧化碳的细胞培养箱中孵育3h,弃去细胞培养基,并用7.4的PBS缓冲液清洗3遍,置于CLSM分别观察4T1细胞与GES-1细胞荧光强度。
如图所示10A所示,将MOF和FA-EM@MOF分别与单核吞噬系统(巨噬细胞)共孵育,通过荧光信号可以看出红色的MOF信号明显高于FA-EM@MOF,说明巨噬细胞对MOF的吞噬效果明显高于FA-EM@MOF。此结果说明,FA-EM 对MOF在血液中的运输具有一定的保护作用,能有效避免被机体的免疫系统清除。而图10B所示,当FA-EM@MOF与正常细胞(GES-1)和肿瘤细胞(4T1)共孵育时,4T1细胞的荧光强度明显高于GES-1细胞,此结果说明,FA-EM修饰的纳米载体能与肿瘤细胞表面的FA受体特异性结合,增强肿瘤细胞对FA-EM 修饰的纳米载体的吞噬,上述实验可以证明本发明制备的FA-EM@GO-MOF和 FA-EM@GO-MOF/DOX可以有效避免被机体的免疫系统清除,增强肿瘤细胞对其吞噬从而增强肿瘤的治疗效果。
Claims (9)
1.一种多功能纳米载体FA-EM@GO-MOF的制备方法,其特征在于,包括如下步骤:
(1)将氧化石墨烯GO与卟啉(TCPP)加入到缓冲液中,超声后干燥得到GO-TCPP储存备用;
(2)将Fe(NO3)3·9H2O和 C2H3NaO2·3H2O加入到有机溶液中,回流反应;离心收集固体沉淀[Fe3O(CH3COO)6(H2O)3](CH3COO);
(3)将[Fe3O(CH3COO)6(H2O)3](CH3COO)与GO-TCPP加入到有机溶液中,并加入甲酸,充分混匀,将此混合溶液高温反应,得到GO-MOF纳米载体;
(4)将DSPE-PEG-FA加入到红细胞溶液中并搅拌,得到DSPE-PEG-FA修饰的红细胞膜FA-EM;
(5)将FA-EM重悬在缓冲液中,并向上述溶液加入步骤(3)的GO-MOF纳米载体搅拌得到FA-EM包裹的GO-MOF(FA-EM@GO-MOF);
其中,步骤(3)中[Fe3O(CH3COO)6(H2O)3](CH3COO)与GO-TCPP的质量比为2-3:1。
2.根据权利要求1所述的多功能纳米载体FA-EM@GO-MOF的制备方法,其特征在于,步骤(1)中GO与TCPP的质量比为1:0.5-1.5。
3.根据权利要求1所述的多功能纳米载体FA-EM@GO-MOF的制备方法,其特征在于,步骤(2)中Fe(NO3)3·9H2O与 C2H3NaO2·3H2O的质量比为1-2:1。
4.根据权利要求1所述的多功能纳米载体FA-EM@GO-MOF的制备方法,其特征在于,步骤(3)中将混合溶液高温反应放入反应釜中,在80-100°C的烘箱中反应20-24 h,得到GO-MOF纳米载体。
5.一种权利要求1所述的制备方法所制备的多功能纳米载体FA-EM@GO-MOF。
6.一种权利要求5所述的多功能纳米载体FA-EM@GO-MOF在制备核磁成像的造影剂、环境响应型载药试剂和光热治疗、光动力治疗试剂中的应用。
7.一种用于肿瘤的治疗药物,其特征在于,所述治疗药物以权利要求1制备的所述的多功能纳米载体FA-EM@GO-MOF为载体并装载抗肿瘤药物。
8.根据权利要求7所述的治疗药物,其特征在于,所述抗肿瘤药物为DOX。
9.一种权利要求7所述的治疗药物的制备方法,其特征在于,包括如下步骤:
(1)将GO与TCPP加入到缓冲液中,超声后干燥,得到GO-TCPP储存备用;
(2)将Fe(NO3)3·9H2O和 C2H3NaO2·3H2O加入到有机溶液中,回流反应;离心收集固体沉淀[Fe3O(CH3COO)6(H2O)3](CH3COO);
(3)将[Fe3O(CH3COO)6(H2O)3](CH3COO)与GO-TCPP加入到有机溶液中,并加入甲酸,充分混匀,将此混合溶液高温反应,得到GO-MOF纳米载体;
(4)GO-MOF溶解于缓冲液中充分混匀,并加入DOX,避光搅拌得到GO-MOF/DOX;
(5)将DSPE-PEG-FA加入到红细胞溶液中并搅拌,清洗,得到DSPE-PEG-FA修饰的红细胞膜FA-EM;
(6)将FA-EM重悬在缓冲液中,并向上述溶液加入GO-MOF/DOX搅拌,得到FA-EM包裹的GO-MOF(FA-EM@GO-MOF/DOX)。
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