CN113230418A - 一种超小核壳结构铁纳米颗粒的制备方法及应用 - Google Patents
一种超小核壳结构铁纳米颗粒的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种超小核壳结构铁纳米颗粒的制备方法及应用。包括如下步骤:(1)超小铁纳米颗粒的合成;(2)多巴胺化聚乙二醇对合成纳米颗粒进行表面修饰;(3)共价键合的方法对上述纳米颗粒修饰肿瘤靶向性穿膜肽iRGD,提高其肿瘤靶向性。以羰基铁为前驱物,油胺为表面配体,十六烷基三甲基溴化铵提供卤素离子,采用油相高温热解法制备了尺寸超小的核壳结构铁纳米颗粒用于肿瘤治疗。本发明合成尺寸超小的核壳结构铁纳米颗粒,具有卓越的稳定性、生物安全性、和抗肿瘤活性。
Description
技术领域
本发明涉及生物医药技术领域,特别涉及壳核结构的超小单晶铁纳米颗粒及其用途。
背景技术
癌症是机体在各种致癌因素的作用下,局部组织中的某个细胞在基因水平上失去对其生长的正常调控,导致其克隆性异常增生大量生长繁殖,局部浸入周围正常组织甚至经由血液***或淋巴***转移到身体其他部位。癌症发病率逐年增高,严重威胁人类的健康。目前常用的治疗方法有手术治疗、化疗、放疗等。其中手术治疗仅适用于早期癌症治疗,应用局限性大;化疗是利用化学药物杀死瘤细胞或抑制其生长繁殖的一种治疗方式。由于化疗药物一般为***给药,因此不可避免地带来全身性毒副作用,如:脱发、贫血、骨髓抑制等;放疗是利用放射性同位素产生的放射线和各类放射线治疗机或加速器产生的放射线治疗恶性肿瘤的方法。往往放疗过程会引起免疫***紊乱、皮肤放射性损伤、乏力、头晕等症状;免疫治疗是指针对机体低下的免疫状态,人为的增强机体的免疫,依靠自身免疫机能杀死癌细胞和肿瘤组织的治疗方法。但治疗过程中往往引起内分泌异常、呕吐、肺炎等,且免疫治疗药物价格昂贵。因此迫切需要开发治疗效果好、毒副作用小、成本低的新型治疗药物和方法。
活性氧(reactive oxygen species,ROS)是机体氧化应激时产生的主要分子,一直以来被认为是肿瘤发生、发展和复发的重要因素。然而近年来研究发现,ROS可以通过诱导肿瘤细胞凋亡、坏死、自噬性细胞死亡实现肿瘤治疗的目的。因此,ROS在肿瘤治疗方面的作用受到越来越多的关注,许多科研工作者对各种增加ROS水平导致细胞发生氧化应激的药物进行了研究,但目前仍然缺乏安全、高效的治疗剂。
铁作为机体中含量最高的微量元素,其安全性是其他各类金属元素无可比拟的。而亚铁离子(Fe2+)具有较高的过氧化氢酶活性,可以促使过氧化氢(H2O2)分解生成高活性的羟基自由基(·OH),这一反应过程被称为芬顿反应(Fenton Reaction)。表达如下:
Fe2++H2O2=Fe3++·OH+HO-(1)
Fe3++H2O2=Fe2++·OOH+H+(2)
这一过程产生的极高活性的羟基自由基可有效的分解多种有机物,多用于污水处理。近年来,科研工作者们发现,肿瘤细胞内及其微环境中存在较高浓度的H2O2,这为芬顿应用的肿瘤治疗提供了契机。同时,铁死亡作为一种区别于细胞凋亡、坏死、自噬的铁依赖性的新型程序性死亡方式受到越来越多的关注。铁死亡是在二价铁或酯氧合酶的作用下,催化细胞膜上高表达的不饱和脂肪酸发生脂质过氧化,从而导致细胞死亡的过程。而铁死亡这一细胞死亡过程被证实和癌症免疫密切相关。联合使用铁死亡致敏剂和免疫检查点抑制剂可产生强烈的免疫反应,进而有益于肿瘤治疗。目前研究较多的是通过递送Fe2+应用于肿瘤治疗。但是Fe2+稳定性差,易被氧化而失去活性,且其本身在肿瘤微环境弱酸性条件下的催化活性有限,很难达到令人满意的治疗效果。因此常需要与其他治疗方式联合应用,进一步增加了治疗难度,不利于临床推广使用。因此如何提高铁纳米颗粒的催化活性是目前研究的重点。
相较于Fe2+,零价铁(Fe(0))具有更强的还原性,因而得到广泛的关注。但是,零价铁的稳定性差是限制其应用的一大关键因素,Fe(0)暴露于空气中很快被氧化为 Fe2+/Fe3 +,极大的降低其催化活性。针对这一问题,目前常做的工作是加入其他元素制备成合金材料提高其稳定性。现有技术中制备了具有多孔黄壳结构的Fe/Fe3O4纳米颗粒,其中铁核大小、Fe3O4壳层厚度及开孔大小均可精确调控。由于氧化铁壳层及憎水空腔层的保护作用,该纳米颗粒在储存条件和生理环境下具有较高的稳定性。而当到达肿瘤微酸环境,其壳层结构快速刻蚀暴露出高活性零价铁核,进而表现出可观的芬顿催化活性和较强的铁死亡活性。
发明内容
发明目的:为了进一步简化超小铁纳米颗粒制备工艺,提高扩大生产及临床应用的可能性。本发明提出一种粒径仅为3-15nm的壳核结构铁纳米颗粒,该纳米颗粒具有极薄的壳层结构和超小的铁核。由于合成过程中加入卤素离子提高了纳米颗粒的稳定性,使其在生理环境不易被氧化,而在肿瘤酸性微环境,超薄的壳层结构快速刻蚀,暴露出尺寸小、比表面积大、表面能高、还原性强的铁核,具有极高的Fenton催化活性,可用于肿瘤细胞的杀伤。同时,该超小铁纳米颗粒可以激起机体免疫,应用于肿瘤的免疫治疗,进一步加强了其抗肿瘤活性。
技术方案:本发明提供超小铁纳米颗粒的制备方法,包括如下步骤:
(1)超小铁纳米颗粒的合成:向十六烷基三甲基溴化铵中加入油胺,升温至 100-120℃并通入惰性气体,保温30-60min以去除反应体系中的水蒸气和氧气,继续升温,加入羰基铁,持续反应20-30min后降至室温,以极性溶剂沉淀纳米颗粒,除去上清液后,沉淀溶于正己烷中,得到超小铁纳米颗粒。
(2)对合成纳米颗粒进行表面修饰:合成超小铁纳米颗粒分散于有机溶剂中,加入多巴胺修饰的聚乙二醇,反应结束后将溶液以惰性气体吹干,得到PEG化的纳米颗粒。
(3)修饰肿瘤靶向性多肽:在上述PEG化的纳米颗粒表面通过共价键修饰具有肿瘤血管内皮细胞靶向性的iRGD多肽,得到具有肿瘤靶向性的纳米颗粒(iRGD-bcc-USINPs)。
进一步地,所述羰基铁为Fe(CO)5。
进一步地,所述核壳结构铁纳米颗粒粒径为3-5nm。
制备得到的PEG化的纳米颗粒用于制备具有抗肿瘤效果的药物。
制备得到的PEG化的纳米颗粒用于制备具有免疫治疗效果的药物。
合成不定形的超小铁纳米颗粒(amor-USINPs)、15nm bcc结晶相的铁纳米颗粒(bcc-FeNPs)和超小四氧化三铁(USIONPs)作为对照。
本发明合成具有超小尺寸的核壳结构铁纳米颗粒,以羰基铁为前驱物,油胺为表面配体,十六烷基三甲基溴化铵提供卤素离子,采用油相高温热解法制备了尺寸超小的核壳结构铁纳米颗粒用于肿瘤治疗。
1、采用高温热解法合成铁纳米颗粒,通过加入十六烷基三甲基溴化铵控制铁纳米颗粒晶型,通过调节合成所用溶剂和温度使纳米颗粒具有3-5nm超小的尺寸。
2、由于卤素离子溴的引入,本发明合成的铁纳米颗粒在生理条件具有良好的稳定性,且其催化活性可以被掩盖。
3、合成结束后,通过对纳米颗粒表面进行配体交换,制备PEG化的纳米颗粒。
4、对PEG末端进行修饰,连接具有肿瘤靶向性的iRGD多肽。
5、该尺寸超小的铁纳米颗粒具有超薄的壳层结构,可以在肿瘤弱酸性条件下快速刻蚀而失去对铁核的保护和活性屏蔽作用。
6、由于比表面积较大,暴露出的超小铁核具有极强的芬顿催化活性,表现出对肿瘤细胞较强的杀伤活性。
7、由于铁纳米颗粒介导的免疫细胞活化,进而表现出对肿瘤细胞具有较强的免疫杀伤效果。
8、本发明制备超高活性铁纳米颗粒,工艺简单,易于扩大生产。
有益效果:本发明制备3-5nm结晶相铁纳米颗粒。该铁纳米颗粒可以在生理条件下稳定存在,不易被氧化。而其超薄的壳层结构在肿瘤酸性条件下快速刻蚀,暴露出铁核。由于铁核具有较大的比较面积,进而表现出极高的芬顿催化活性,具有较强的肿瘤杀伤作用和免疫治疗效果。
附图说明
图1为纳米颗粒合成及作用机制示意图:(A)bcc-USINPs铁释放及其催化羟基自由基生成的示意图;(B)肾脏可清除的超小单晶铁纳米颗粒介导高效可控的铁死亡促进免疫治疗的体内机制示意图;
图2为纳米颗粒的透射电镜表征图:(A)bcc-FeNPs;(B)USIONPs;(C)bcc-USINPs;(D)amor-USINPs;
图3为所合成的不同结晶度的超小铁纳米颗粒在刚合成和合成后7天的TEM图像:(A)bcc-USINPs合成后立即观察得到的高分辨TEM图像;(B)bcc-USINPs纳米颗粒在空气中暴露7天后的透射电镜表征图;(C)amor-USINPs合成后立即观察得到的高分辨 TEM图像;(D)amor-USINPs纳米颗粒在空气中暴露7天后的透射电镜表征图;
图4为纳米颗粒的磁学性质表征图:(A)bcc-USINPs在合成后24h内的磁学性质表征图;(B)amor-USINPs在合成后24h内的磁学性质变化表征图;
图5为不同纳米颗粒的X射线衍射图;
图6为上述四种纳米颗粒在pH 7.4和pH 5.5缓冲溶液中的铁释放情况结果图;
图7为bcc-FeNPs、USIONPs和amor-USINPs纳米颗粒在pH 5.5条件下催化1mM 过氧化氢产生羟基自由基用于亚甲基蓝降解的结果图;
图8为超小铁纳米颗粒在pH 7.4和pH 5.5条件下催化1.0mM过氧化氢产生羟基自由基用于亚甲基蓝降解的结果图(A);以及双氧水浓度(0-1mM)对该降解作用效率的影响(B);
图9为上述四种纳米颗粒催化过氧化氢分解的米氏常数:(A和B)bcc-USINPs;(C和D)amor-USINPs;(E和F)15nm bcc-Fe NPs;(G和H)USIONPs的Michaelis-Menten 动力学和lineweaverer-burk绘图;(I)上述各纳米颗粒KM和Vmax计算结果;
图10为上述纳米颗粒作用后人肝癌细胞HepG2产生活性氧簇的情况;
图11为上述各铁纳米颗粒对人肝癌细胞HepG2的毒性(A);bcc-USINPs和 amor-USINPs纳米颗粒合成7天后对HepG2细胞的毒性(B);
图12为上述各种铁纳米颗粒对HepG2细胞线粒体膜电位的影响;
图13为细胞与bcc-USINPs共孵育4h后生物电镜的结果图:(a)单个细胞全貌;(b和c)对细胞局部放大的TEM图;
图14为bcc-USINPs作用后细胞膜表面钙网蛋白的表达情况;
图15为MC38细胞经bcc-USINPs处理后对DC细胞成熟的影响;
图16为荧光标记的铁纳米颗粒通过尾静脉注射进入小鼠体内后的分布情况:(A)HepG2荷瘤小鼠在静脉注射bcc-USINPs或iRGD-bcc-USINPs后0、2、4、8、12和24h 的体内荧光图像;(B)注射后24小时对主要器官和肿瘤进行体外荧光图像;
图17为iRGD-bcc-USINPs纳米颗粒对HepG2荷瘤小鼠的体内抗肿瘤实验结果:(A)体内抗肿瘤试验方案示意图;(B)不同给药方式后荷瘤小鼠的肿瘤生成曲线;(C)不同给药方式对荷瘤小鼠的生存期影响;(D)观察期结束后各组小鼠进行解剖,对得到的肿瘤拍照;(E)铁纳米颗粒治疗后小鼠瘤重的变化;(F)iRGD-bcc-USINPs纳米颗粒作用后的肿瘤的H&E结果图;(G)iRGD-bcc-USINPs纳米颗粒作用后的肿瘤的TUNEL结果图; (H)纳米颗粒治疗后小鼠体重变化结果图;
图18为MC38荷瘤小鼠的体内免疫治疗实验结果:(A)小鼠免疫治疗实验方案示意图;(B)不同给药方式后荷瘤小鼠的肿瘤生成曲线;(C)不同给药方式对荷瘤小鼠的生存期的影响;(D)各组荷瘤小鼠第二次接种肿瘤的生长曲线;
图19为MC38荷瘤小鼠体内免疫检测结果:(A)不同治疗方式对小鼠***中DC 成熟影响的流式分析及成熟DC含量统计;(B)不同治疗方式对小鼠肿瘤浸润T细胞影响的流式分析和杀伤性T细胞(CD8+T细胞)含量统计;(C)不同治疗方式对小鼠肿瘤中Treg细胞影响的流式分析及占CD4+T细胞的含量统计;
图20为iRGD-bcc-USINPs纳米颗粒体内安全性评价结果:(A、B、C、D、E、F)小鼠血常规检测;(G、H、I、J)小鼠血生化指标检测;(K)各主要组织器官的H&E染色;
本发明所使用的五羰基铁(Fe(CO)5)、油胺(OAm,70%)、十六烷基三甲基溴化铵(CTAB,98%)、亚甲基蓝(MB)、N-羟基琥珀酰亚胺(NHS,98%)、N-(3-二甲基氨基丙基)- 乙基二亚胺盐酸盐(EDCI,98%)、甲氧基(聚乙二醇)多巴胺(mPEG-DPA,Mw=2000)、Cy3 和Cy7,2′,7′-二氯二氢荧光素(DCFH-DA)和5,5,6,6-四氯-1,1,3,3-四乙基苯亚咪唑基碳菁氯(JC-1)均可购买获得。
所用人肝癌HepG2细胞系和小鼠结肠癌MC38细胞系均取自ATCC(American TypeCulture Collection),在37℃、5%CO2湿化培养箱中以10%胎牛血清和1%青霉素/链霉素的DMEM培养基培养,每隔一天传代一次。
BALB/cu裸小鼠(18-22g)和C57BL/6小鼠维持饲养于适当的条件下。所有动物实验均按照《实验动物护理和使用指南》进行。
实施例1:铁纳米颗粒制备方法:
结晶相超小铁纳米颗粒(bcc-USINPs)的合成:精确称量80mg CTAB置于四颈瓶中,加入OAm(15mL,45mmol),升温至120℃,持续通入氩气以去除体系中的水蒸气和氧气,30min后升温至260℃,注入Fe(CO)5(0.05mL,0.37mmol),保温30min,然后冷却到室温。加入乙醇沉淀出纳米颗粒,10000rpm离心5min,随后弃去上清,沉淀重新溶解于正己烷中,反复清洗三遍,得到结晶相的超小铁纳米颗粒。
不定形超小铁纳米颗粒(amor-USINPs)的合成:不定形超小铁除了在合成过程中不加入CTAB,其余操作与上述bcc-USINPs一致。
15nm铁纳米颗粒(bcc-FeNPs)的合成:精确称取0.15g溴化铵置于四颈瓶中,加入ODE(20mL)和OAm(0.3mL,0.9mmol),120℃下持续通入氩气以除去体系用的氧气和水蒸气,30min后以10℃/min升温至180℃,通过注射器注入Fe(CO)5(0.7mL,5.2mmol)。反应体系在180℃下保持20分钟,然后冷却到室温。以乙醇沉淀颗粒,8000rpm离心5 min后,弃去上清,沉淀溶于正己烷中,反复清洗三遍,收集颗粒备用。
3nm四氧化三铁纳米颗粒(USIONPs)的合成:称取1.8g油酸铁复合物,0.57g油酸,1.61g油醇置于四颈瓶中,加入10g二苄醚以溶解上述各成分。室温下通入氩气去除反应体系中的氧气,随后以10℃/min升温至250℃,并在此温度保持30min。反应结束后以乙醇沉淀颗粒,8000rpm离心5min弃去上清,收集颗粒溶解于正己烷中备用。
实施例2:纳米颗粒的基础表征。
采用透射电镜(TEM)对上述纳米颗粒进行观察(图2):A图所示为bcc-FeNPs的TEM图像;B图为3nm USIONPs的TEM图像,C图为bcc-USINPs合成后即刻观察的TEM 图;D图为amor-USINPs合成后即刻观察的TEM图。为了验证bcc-USINPs的稳定性,放置7天后再次对其形貌进行观察。如图3A、B,暴露于空气中7天后bcc-USINPs的高分辨TEM并未发生显著变化。图3C、D中,放置7天后,amor-USINPs的铁核被氧化而几乎消失。反映出bcc-USINPs具有较好的稳定性。
采用高灵敏度振动样品磁强计(VSM)对超小铁纳米颗粒合成后放置24h期间的磁学性质进行了评价。观察发现,bcc-USINPs的饱和磁化强度显著强于amor-USINPs。并且,bcc-USINPs的饱和磁化强度在24h内下降了16.5%(图4A),而amor-USINPs在相同时间内下降了50.4%(图4B),表明bcc-USINPs的稳定性远高于amor-USINPs。
图5为X射线衍射(XRD)对各颗粒的晶型进行分析,其中bcc-FeNPs可以观察到明显的Fe3O4和bcc-Fe衍生峰;相较于不定形超小铁,bcc-USINPs的XRD结果图中可以观察到Fe3O4和bcc-Fe衍生峰。
实施例3:各超纳米颗粒的表面修饰。
基于邻二酚羟基对铁具有配位作用力,所以本发明中采用多巴胺修饰的聚乙二醇对铁纳米颗粒表面进行修饰。具体制备过程如下:
称取各纳米颗粒25mg,置于样品瓶中,加入100mg甲氧基聚乙二醇-多巴胺 (mPEG-DPA,Mw=2000Da),量取4mL氯仿溶解上述固体。置于摇床震荡反应4h,反应结束后对纳米颗粒进行干燥,去除氯仿,随后加入2mL超纯水复溶。使用PD-10 柱子纯化所得纳米颗粒,即得到PEG修饰的纳米颗粒。
为提高纳米颗粒的肿瘤靶向性,进一步地对其表面修饰iRGD多肽,实现纳米颗粒的肿瘤靶向。其具体操作过程为:精确称取纳米颗粒25mg,置于样品瓶中,加入90mg 甲氧基聚乙二醇-多巴胺(mPEG-DPA,Mw=2000Da),10mg马来酰亚胺聚乙二醇-多巴胺(MAL-PEG-DPA,Mw=2000Da),量取4mL氯仿溶解上述固体。随后振荡反应4h,反应结束后对纳米颗粒进行干燥,得到PEG化的纳米颗粒。向干燥后的纳米颗粒中加入2mg肿瘤靶向多肽iRGD-SH,继续反应4h。反应结束后使用PD-10柱子纯化所得纳米颗粒,即得到具有肿瘤靶向性的纳米颗粒。
本实施例中,采用配体交换的方法,对所合成纳米颗粒表面进行修饰,使油相合成的超小铁纳米颗粒转至水溶液,从而拥有良好的生物相容性和稳定性。进一步地对其表面修饰具有肿瘤新生血管内皮细胞过度表达αvβ3整合素识别能力的iRGD多肽,实现纳米颗粒的肿瘤靶向性。通过上述修饰进一步地提高了纳米颗粒的肿瘤治疗能力。
实施例4:pH依赖性铁离子释放。
为了确保上述合成的铁纳米颗粒可以在循环***中保持稳定而在肿瘤酸性微酸环境中释放出铁离子发挥抗肿瘤作用,我们对其在不同pH缓冲溶液中的铁释放能力做了验证。具体操作如下:
精确量取1mL各铁纳米颗粒(2mg/mL)置于Mw=500的透析袋中,将透析袋浸没于50mL pH 7.4或pH 5.0的缓冲溶液中,于相应时间点取出1mL缓冲溶液并补加1mL 相性酸度的空白缓冲溶液。取样结束后用电感耦合等离子色谱测定各样品中的铁含量,进而计算出铁释放量。计算公式如下:
Rt=Ct x 50/2x 100%
其中Rt是响应时间点的铁释放率,Ct是该时间点取出溶液中的铁浓度,单位为:mg/mL。
随后通过释放率对时间作图,得到图6。可以观察到,bcc-USINPs在pH 7.4仅具有微弱的铁释放,而在pH 5.0缓冲溶液中的铁释放率可超过30%。其他各形式纳米颗粒即便在pH 5.5的缓冲溶液中的铁释放率均低于5%。
实施例5:溶液中芬顿催化活性测定。
为了验证铁纳米颗粒的芬顿催化活性,采用亚甲基蓝(MB)降解实验检测铁纳米颗粒催化双氧水分解产生·OH的能力。具体操作如下:
精密量取60μL上述各铁纳米颗粒(1mg/mL,60μg)加入含有1mM H2O2的亚甲基蓝溶液(10mg/L),整个反应控制在不同pH(pH=7.4和pH=5.5)下反应30min,并维持反应温度为40℃。反应结束后将上述溶液离心,以去除残留纳米颗粒的影响。使用紫外 -可见光谱(MultiskanSky,Thermo Fisher Scientific)测定上清液的吸收光谱。如图7所示, amor-USINPs、15nm FeNPs和USIONPs纳米颗粒均未对MB产生明显的降解,这主要是因为各纳米颗粒铁释放量较低。如图8A所示,bcc结晶相的超小铁在pH 5.5显示出对MB极强的降解能力,而其在pH 7.4条件下表现出的MB降解能力远低于酸性条件下。表明bcc-USINPs催化过氧化氢分解产生羟基自由基的活性与pH有关。再次验证了 bcc-USINPs具有酸激活的过氧化氢酶活性。并且,该降解活性与溶液中的H2O2浓度呈正相关(图8B)。
进一步地,我们对上述各铁纳米颗粒降解MB的米氏常数(KM)进行了计算(图9)。可以观察到,bcc-USINPs的Km为0.31mM,远低于amor-USINPs、bcc-FeNPs和 USIONPs。
实施例6:细胞水平产生活性氧能力的测定。
采用2′,7′-二氯二氢荧光素(DCFH-DA)对纳米颗粒作用后的细胞进行染色,以验证上述各铁纳米颗粒在细胞水平产生活性氧的水平。选择人肝癌细胞HepG2接种于48孔板中,培养过夜。向细胞培养液中加入各纳米颗粒使其终浓度为20μg/mL,37℃下孵育 4h。孵育完成后,移除含有铁纳米颗粒的细胞培养液,加入DCFH-DA对细胞进行染色。随后,使用荧光显微镜对染色后的细胞进行观察。如图10所示,可观察到bcc-USINPs 可以显著提高细胞的ROS水平,有较强的绿色荧光。
实施例7:超小铁纳米颗粒的细胞毒性测定。
HepG2细胞接种于96孔板中,培养过夜。当细胞生长至70%汇合度时加入各铁纳米颗粒(3-100μg/mL),培养24h后移除含有纳米颗粒的培养基,加入CCK-8置于37℃培养箱继续培养30min,使用酶标仪测量在405nm的吸光值,计算细胞存活率。如图 11A所示,15nmbcc-FeNPs和USIONPs测试浓度范围内未表现出明显的肿瘤细胞杀伤活性。结晶相和不定形超小铁纳米颗粒均表现出较高的细胞毒性,其IC50大约为20 μg/mL。值得注意的是,在放置7天后结晶相超小铁的细胞毒性保持相对稳定,而 amor-USINPs毒性显著下降,这一现象再次证明了bcc-USINPs卓越的稳定性(图11B)。
实施例8:超小铁纳米颗粒对细胞线粒体功能的影响。
上述结果表明,bcc-USINPs可在细胞中催化产生大量ROS,而线粒体作为细胞的供能场所,对氧化性物质尤为敏感。因为,我们进一步研究了超小铁纳米颗粒对细胞线粒体功能的影响。本实施例采用5,5,6,6-四氯-1,1,3,3-四乙基苯亚咪唑基碳菁氯(JC-1) 对各纳米颗粒作用后的细胞进行染色。当线粒体完整、膜电位较高时,JC-1聚集在线粒体的基质中,形成聚合物,产生红色荧光;在线粒体膜电位较低时,JC-1不能聚集在线粒体的基质中,此时JC-1为单体,可以产生绿色荧光。如图12,bcc-USINPs作用后的细胞绿色荧光显著增强,提示其对肿瘤细胞线粒体存在破坏。
实施例9:bcc-USINPs对细胞形貌的影响。
线粒体结构和功能的改变往往会导致细胞形态的变化。因此,本实施例采用生物电镜对bcc-USINPs作用后的小鼠结肠癌细胞MC38进行观察。如图13,可以观察到细胞完整性受到破坏,形成较多碎片。纳米颗粒作用后的细胞线粒体膜结构消失,线粒体嵴破坏。
实施例10:bcc-USINPs诱导肿瘤细胞膜表达钙网蛋白。
细胞膜表面钙网蛋白(CRT)的表达水平是免疫激活和治疗效果的重要指标,因此本实施例通过激光共聚焦实验检测bcc-USINPs处理后肿瘤细胞表面CRT的表达情况。选择小鼠结肠癌细胞MC38接种于共聚焦皿中,培养过夜。向细胞培养液中加bcc-USINPs (5,10,20μg/mL),37℃下孵育4h。孵育完成后,移除含有铁纳米颗粒的细胞培养液,对细胞进行封闭,随后加入CRT荧光抗体进行染色。最后,使用共聚焦对细胞进行观察。如图14,bcc-USINPs作用后的细胞膜表面有较强的荧光,表明肿瘤细胞在该纳米颗粒的作用下有较高的CRT膜表达量。
实施例11:bcc-USINPs促进树突状细胞成熟的作用。
CRT蛋白在肿瘤细胞膜表面表达量提高,可以发送“eat me”的信号并刺激树突状细胞(DC)吞噬这些癌细胞。为了验证bcc-USINPs处理后的肿瘤细胞可以诱导DC的成熟,本实施例收集上述肿瘤细胞的培养液对DC进行孵育,随后采用流式细胞仪测试DC细胞的成熟率。如图15,相较于未处理的DC,20μg/mL bcc-USINPs预孵育的癌细胞培养液处理后的DC成熟率提高了7.5倍。
实施例12:荧光标记的bcc-USINPs颗粒的体内分布。
取上述制备所得的iRGD靶向肽修饰及未修饰的PEG化的超小铁纳米颗粒,进一步地通过酰胺反应共价标记荧光染料Cy7,标记完成后透析去除游离荧光染料。小鼠接种HepG2肿瘤生长至200mm3后随机分为两组(n=3)。通过尾静脉注射标记荧光染料的超小铁纳米颗粒进入小鼠体内(注射剂量为Cy7:0.5mg/kg),通过小动物活体成像仪(柯达多模成像***IS2000MM)对超小铁纳米颗粒在小鼠体内的分布进行观察。如图16所示, iRGD靶向肽修饰的超小铁纳米颗粒相较于未修饰的颗粒具有显著提高的肿瘤靶向性和滞留能力,为进一步的抗肿瘤治疗提供了必不可少的条件。
实施例13:HepG2荷瘤小鼠的体内治疗效果。
接种HepG2肿瘤细胞后的荷瘤鼠随机分为四组:生理盐水组、高浓度 iRGD-bcc-USINPs(10mg/kg)尾静脉给药一次、低浓度iRGD-bcc-USINPs(1mg/kg)尾静脉给药1次、低浓度iRGD-bcc-USINPs给药3次组(1mg/kg)(图17A),各组小鼠给药后每隔一天量一次肿瘤体积。如图17B所示,低剂量多次给药组中的肿瘤得到明显抑制,甚至出现消融。同时给药后荷瘤小鼠的生存期得到了极大的延长,在为期50天的观察中低浓度给药三次的小鼠未出现死亡现象(图17C)。因此,本实施例确定最终给药方式为以1mg/kg的剂量每三天静脉注射iRGD-bcc-USINPs一次,共注射三次。观察期结束后,对剩余小鼠进行解剖,称量各组小鼠瘤重。如图17D,E所示,经过多次低计量 iRGD-bcc-USINPs治疗后的小鼠的肿瘤体积和重量显著下降。同时对解剖的肿瘤进行H&E和TUNEL染色,可以观察到超小铁纳米颗粒治疗后的肿瘤有显著的凋亡和坏死,再次证明了bcc结晶相超小铁卓越的抗肿瘤效果(图17F,G)。在整个治疗过程中,小鼠的体重未出现明显下降,表明纳米颗粒有较好的生物相容性(图17H)。
实施例14:结肠癌MC38荷瘤小鼠抗肿瘤治疗及肿瘤长期免疫考察。
为了研究iRGD-bcc-USINPs的体内肿瘤免疫治疗效果,本实施例建立了小鼠结肠癌 MC38皮下荷瘤鼠模型。随机分为四组:生理盐水组、腹腔注射aPD-L1(45μg/mouse)、静脉注射iRGD-bcc-USINPs(1mg/kg)、静脉注射iRGD-bcc-USINPs 24h后腹腔注射 aPD-L1(iRGD-bcc-USINPs:1mg/kg,aPD-L1:45μg/mouse)。各组小鼠每三天给药一次,共给药三次,期间每隔一天量一次肿瘤体积(图18A)。如图18B所示,联合aPD-L1免疫检查点阻断后,iRGD-bcc-USINPs给药组小鼠肿瘤体积呈现显著抑制。相较于生理盐水组和腹腔注射aPD-L1组,单独给予iRGD-bcc-USINPs和联合使用aPD-L1后小鼠生存期延长两倍以上(图18C)。
为了评估治疗后小鼠肿瘤长期免疫效果,本实施例建立了肿瘤再挑战模型。在首次接种小鼠结肠癌MC38皮下荷瘤后,分别给予生理盐水、aPD-L1(45μg/mouse)、 iRGD-bcc-USINPs(1mg/kg)、iRGD-bcc-USINPs+αPD-L1(iRGD-bcc-USINPs:1mg/kg,αPD-L1:45μg/mouse)。治疗三次后,荷瘤小鼠进行第二次皮下瘤接种。随后对各组小鼠进行持续观察,记录第二次接种肿瘤生长情况,但不给予额外的治疗(图18A)。如图 18D,相较于未治疗组和单独αPD-L1,iRGD-bcc-USINPs组小鼠的继发性肿瘤生长明显更加缓慢。尤其当纳米颗粒联合免疫检查点抑制剂αPD-L1后,80%的小鼠可继续保持无肿瘤的状态,这表明它们具有长期的抗癌免疫力。
实施例15:结肠癌MC38荷瘤小鼠的体内治疗微环境检测。
为了研究纳米颗粒bcc-USINPs的体内抗肿瘤免疫激活机制,本实施例建立小鼠结肠癌MC38皮下荷瘤鼠模型,并在第三次给药后,收集各组肿瘤和***用于进一步检测分析。针对肿瘤组织微环境中的免疫激活检测,我们首先将收集好的肿瘤组织,放入 6孔板中,之后加入含有胶原酶的DMEM无血清培养基,并用眼科剪将肿瘤组织剪碎,随后在37℃培养箱中消化肿瘤组织30分钟。然后将孔板取出,吸取消化后的组织液在 200目筛网上对肿瘤组织进一步研磨处理,收集经过筛网研磨后的滤液。然后在600g 条件下离心15分钟,弃掉上清后,收集底部的肿瘤细胞以备后续流式染色处理。同时针对收集的肿瘤***组织,采用跟肿瘤组织相同的剪碎,消化处理方式得到细胞悬液后,用PBS清洗一遍后以备流式染色处理。
为了检测给药后小鼠***中树突状细胞成熟情况,将收集的淋巴细胞与区分细胞死活的染料FVS 780,抗CD45-FITC,抗CD11c-APC和抗MHC-cy5.5,抗CD80-BV421,抗CD86-PE混合孵育50分钟,每隔10分钟涡旋混匀一次,之后用PBS洗涤两次,并根据标准方案通过流式细胞术进行检测。实施例10中已经证明iRGD-bcc-USINPs给药后可以增加CRT表达,CRT发送“eat-me”信号并刺激树突状细胞来吞噬这些癌细胞。如图19A,本实施例中iRGD-bcc-USINPs作用后树突状细胞的成熟增强。并且这一效应在联合使用免疫检查点抑制剂αPD-L1后进一步提高,为效应T细胞的激活和肿瘤细胞杀伤提供了可能。
足够的T细胞浸润是提高免疫治疗的决定因素之一。为了检测肿瘤组织中浸润的T细胞和Tregs,将收集肿瘤细胞与死活染料FVS 780,抗CD45-FITC,抗CD3-Percp-cy5.5,抗CD8-PE-Cy7,抗CD4-APC,抗CD25-BV421混合孵育50分钟,之后用PBS清洗一遍细胞,然后再用细胞固定破膜试剂盒中的缓冲液清洗一遍细胞,之后用涡旋仪使细胞分散均与,加入Fixation/permeabilization工作液固定细胞约45分钟,每间隔10分钟涡旋一次。然后在每个离心管中加入2mL 1×permeabilization缓冲液,在300g条件下室温离心5分钟,弃去上清,用PBS清洗一遍后,加入抗Foxp3-PE孵育50分钟,之后用PBS清洗两遍,并根据标准方案通过流式细胞仪(BD 453FASCVerse,美国)对各个免疫指标进行检测。如图19B,与未治疗组相比,iRGD-bcc-USINPs通过诱导免疫原性细胞死亡,提高了CD8+T和CD4+T细胞在肿瘤的浸润。并且在联合在aPD-L1后观察到CD8+T细胞明显多于iRGD-bcc-USINPs组,表明阻断PD-L1有效地增加了T细胞的浸润。
同时,Foxp3+Tregs在肿瘤微环境中表现出免疫抑制作用,可以抑制或下调CD8+T细胞的诱导和增殖。如图19C,iRGD-bcc-USINPs可以抑制肿瘤微环境中的Tregs,使其从未治疗组的32.7%降低到约17.6%,而这一含量在联合PD-L1治疗后进一步降低至9.45%,这证明我们的纳米颗粒可通过抑制肿瘤微环境中的负性免疫调节细胞Tregs 更好地消除免疫抑制作用来增强抗PD-L1免疫治疗。
实施例16:iRGD-bcc-USINPs生物安全性评价。
正常小鼠随机分为两组:生理盐水组、iRGD-bcc-USINPs(10mg/kg)组。每组小鼠每三天给药一次,共给药三次。最后一次给药后三天取各组小鼠全血分析其血常规指标,血浆分析血生化指标,并通过H&E染色观察各主要组织器官(心、肝、脾、肺、肾)病理学改变(图20)。
Claims (5)
1.一种超小核壳结构铁纳米颗粒的制备方法,其特征在于:包括如下步骤:
(1)超小铁纳米颗粒的合成:向十六烷基三甲基溴化铵中加入油胺,升温至100-120℃并通入惰性气体,保温30-60min以去除反应体系中的水蒸气和氧气,继续升温,加入羰基铁,持续反应20-30min后降至室温,以极性溶剂沉淀纳米颗粒,除去上清液后,沉淀溶于正己烷中,得到超小铁纳米颗粒。
(2)对合成纳米颗粒进行表面修饰:合成超小铁纳米颗粒分散于有机溶剂中,加入多巴胺修饰的聚乙二醇,反应结束后将溶液以惰性气体吹干,得到PEG化的纳米颗粒。
(3)修饰肿瘤靶向性多肽:在上述PEG化的纳米颗粒表面通过共价键修饰具有肿瘤血管内皮细胞靶向性的iRGD多肽,得到具有肿瘤靶向性的纳米颗粒(iRGD-bcc-USINPs)。
2.根据权利要求1所述的超小核壳结构铁纳米颗粒的制备方法,其特征在于:所述羰基铁为Fe(CO)5。
3.根据权利要求1所述的超小核壳结构铁纳米颗粒的制备方法,其特征在于:所述核壳结构铁纳米颗粒粒径为3-5nm。
4.权利要求1制备得到的PEG化的纳米颗粒用于制备具有抗肿瘤效果的药物。
5.权利要求1制备得到的PEG化的纳米颗粒用于制备具有免疫治疗效果的药物。
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