CN111671769B - 含铜铁氧体纳米粒子在制备抗肿瘤药物中的应用 - Google Patents
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Abstract
Description
技术领域:
本发明属于纳米材料医学领域,具体涉及一种含铜铁氧体纳米粒子在制备抗肿瘤药物中的应用。
背景技术:
铁死亡(Ferroptosis)是一种新发现的、由铁介导的调节性细胞死亡方式。与凋亡、坏死和自噬不同,铁死亡以铁离子依赖的ROS过度积累和脂质过氧化物(LipidPeroxidation,LPO)堆积为特征,可通过添加过量的铁离子充分诱导。
体循环中的铁通常以稳定的三价铁(Fe3+)形式存在与转运。Fe3+与转铁蛋白结合后可以通过转铁蛋白受体1(Transferrin Receptor 1,TFR1)进入细胞,储存于内涵体(Endosome)。内涵体的铁氧化还原酶(Six Transmembrane Epithelial Antigen of theProstate3,STEAP3)可将Fe3+还原为不稳定的、具有催化活性的二价铁(Fe2+)。过量的Fe2+可经二价金属转运体1(Divalent Metal Transporter 1,DMT1)从内涵体释放到细胞浆,存储于铁蛋白(Ferritin)中形成不稳定铁池。当Fe2+过载超出铁蛋白缓冲能力时,溶酶体将使之降解,释放出的Fe2+将通过芬顿反应生成大量的羟基自由基(·OH,最具氧化活性的ROS)。当ROS产生的量超过细胞抗氧化系统的清除能力时,将导致LPO堆积,细胞铁死亡。因此,利用铁离子通过芬顿反应诱导铁死亡成为当今抗肿瘤研究的新方向。
芬顿(Fenton)反应或芬顿样反应作为生物体内OH-(活性最强的ROS之一)的主要来源,是诱导细胞铁死亡的重要途径。由于纳米材料具有向肿瘤组织的特性,细胞酸性溶酶体又能够使之水解并释放纳米递送体系中的Fe2+,因此近年出现了大量以芬顿反应诱导铁死亡为目标的纳米载铁递送体系,其中以四氧化三铁纳米粒(Fe3O4 NPs)的研究最为广泛。然而,研究显示,单纯纳米铁递送体系不能有效诱导肿瘤细胞铁死亡。这是因为Fe3O4NPs中Fe2+和Fe3+之间的间隔电荷可能阻碍了其氧化还原活性,并因此降低了Fe2+的催化活性,最终将导致芬顿反应效率低下,以致于其生成的ROS不足以引发铁死亡。因此如何克服一般芬顿反应局限性、增加细胞铁死亡率,是当今该领域研究的科学难题。
发明内容:
本发明的第一个目的是提供含铜铁氧体纳米粒子在制备抗肿瘤药物中的应用。
本发明的含铜铁氧体纳米粒子,其结构式为Cu(II) xFe(II) 1-xFe(III) 2O4,其中1≥X≥0.5。
本发明进一步通过将Fe3O4纳米粒子中1/2的二价铁(Fe2+)用二价铜(Cu2+)进行取代,合成了新的含铜铁氧体纳米粒子Cu0.5Fe(II) 0.5Fe(III) 2O4(以下简称为Cu@Fe NPs),从而对在Fe3O4纳米粒子中的的电子耦合作用进行消除。所获得的Cu0.5Fe(II) 0.5Fe(III) 2O4纳米粒子具有非常优良的抗肿瘤作用,由此,优选所述的含铜铁氧体纳米粒子,其结构式为Cu0.5Fe(II) 0.5Fe(III) 2O4。
所述的含铜铁氧体纳米粒子是通过以下方法制备的,其是将Fe3O4纳米粒子中X的二价铁用二价铜进行取代,得到含铜铁氧体纳米粒子,其中1≥X≥0.5。
优选,将Fe2+和Gu2+按照物质的量之比(1-X):X加入到水中、并加入过量的Fe3+,以及聚乙烯吡咯酮,制得铜铁前驱体溶液,加热条件下,再加入含氢氧化钠和聚乙烯吡咯酮的水溶液,反应生成含铜铁氧体纳米粒子。
进一步优选,是将CuCl2、FeCl2、FeCl3溶于水中,使得Cu2+,Fe2+,Fe3+物质的量之比为1:1:4,加入聚乙烯吡咯酮,制得铜铁前驱体溶液,将铜铁前驱体溶液加热至85℃至90℃,然后滴加预热至85℃至90℃的含氢氧化钠和聚乙烯吡咯酮的水溶液进行反应,反应后取出固体产物,依次用水、乙醇、丙酮和水洗涤,获得洗涤后的产物,即为含铜铁氧体纳米粒子Cu0.5Fe(II) 0.5Fe(III) 2O4。
进一步优选,所述的铜铁前驱体溶液,其含0.1mM的CuCl2,0.1mM的FeCl2,0.4mM的FeCl3和5mg/ml的聚乙烯吡咯酮,所述的含氢氧化钠和聚乙烯吡咯酮的水溶液是含1.6mM的NaOH和5mg/ml的聚乙烯吡咯酮;将铜铁前驱体溶液加热至85℃至90℃,然后滴加预热至85℃至90℃的含氢氧化钠和聚乙烯吡咯酮的水溶液进行反应,反应后取出固体产物,依次用水、乙醇、丙酮和水洗涤,获得洗涤后的产物。
本发明的第二个目的是提供一种抗肿瘤药物,其含有上述含铜铁氧体纳米粒子作为活性成分。
所述的抗肿瘤药物是抗乳腺癌或卵巢癌的药物。
附图说明:
图1是含铜铁氧体纳米粒子Cu@Fe NPs反应装置的组成图,A:三颈圆底烧瓶;B:冷凝管;C:温度计;D:注射器;
图2是Fe3O4 NPs和Cu@Fe NPs的X射线衍射分析图;
图3是(A)为Cu@Fe NPs的3D结构图,蓝球(图中的1):Cu2+/Fe2+;棕球(图中的2):Fe3 +;白球:O;(B)为Cu@Fe NPs的透射电镜图(50μM);(C)为Cu@Fe NPs的动态光散射图(DLS);
图4是Cu@Fe NPs对卵巢癌A2780细胞ROS生成的影响(72h)。
具体实施方式:
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1:
1、本发明的含铜铁氧体纳米粒子Cu@Fe NPs的制备与鉴定。
将CuCl2、FeCl2、FeCl3溶于100ml水中,使得浓度为0.1mM的CuCl2,0.1mM的FeCl2,0.4mM的FeCl3,并加入500mg聚乙烯吡咯酮(K40),混合均匀得到Cu2+,Fe2+,Fe3+摩尔比为1:1:4的铜铁前驱体溶液。将NaOH溶解于装有100mL去离子水的三颈圆底烧瓶中,使NaOH浓度为1.6mM,并加入500mg聚乙烯吡咯酮(K40),保持在800rpm~1200rpm的转速下混合均匀,在油浴中加热至85℃至90℃。按照图1所示在铁架台上组装冷凝管B、烧瓶A、温度计C和注射器D,将铜铁前驱体溶液放入冷凝管下的烧瓶中。当NaOH溶液温度达到85℃至90℃时,使用注射器以3.5mL/min的速度滴加铜铁前驱体溶液,反应30min后,取出产物,在转速为5000rpm的条件下后,离心产物依次经5mL水、5mL乙醇、5mL丙酮和5mL水离心洗涤,最后得到洗涤后的产物(即为Cu0.5Fe(II) 0.5Fe(III) 2O4纳米粒子,命名为Cu@Fe NPs),将洗涤后的产物分散于水中,得到Cu0.5Fe(II) 0.5Fe(III) 2O4纳米粒子溶液。经X射线衍射分析显示,Cu@Fe NPs为物相组分单一且纯度高的纳米粒子,无其他杂质物相组分(图2)。经透射电镜(TEM)和动态光散射(DLS)检测,Cu@Fe NPs的粒径为10nm至60nm(图3)。
2、本发明的Cu@Fe NPs的抗肿瘤作用
体外培养MDA-MB-231、A2780、A2780cis、CAOV-3和SKOV3以及A549细胞。利用噻唑蓝(MTT)法,研究Cu@Fe NPs对多种肿瘤细胞的作用。
实验方案:上述细胞以1×104/孔的数量种96孔板,加DMEM培养液,放于含5%CO2,的37℃培养箱培养,待细胞贴壁后,每孔加入100μL不同浓度的Cu@Fe NP(0.04μM-20μM),对照组加等体水。Cu@Fe NPs处理72h后,每孔加入10μL MTT溶液,继续培养2h后,加入二甲基亚砜(DMSO),再用酶联免疫检测仪检测540nm的光吸收值,与对照组相比,研究肿瘤细胞增殖情况,利用Excel的FORECAST命令计算IC50。
结果显示,与顺铂相比较,Cu@Fe NPs对体外培养的三阴性乳腺癌MDA-MB-231细胞、卵巢癌细胞如A2780、A2780cis、CAOV-3和SKOV3细胞以及肺癌A549细胞均具有强大的抑制作用(表1)。
表1 Cu@Fe NPs对体外培养肿瘤细胞抑制作用的IC50(CCK8法,72h)
3、Cu@Fe3O4 NP对活性氧的诱发作用。
实验方案:A2780细胞以2×104/孔的数量接种于96孔板中,细胞贴壁后吸弃培养液,参照加入不同浓度的Cu@Fe NPs,继续培养72h,用PBS洗涤2次。以DCFH-DA为检测细胞内活性氧的水平荧光探针,每孔加入100μL DCFH-DA(25μM),于37℃染色45分钟,然后再次用PBS洗涤2次,酶标仪检测细胞荧光强度(激发=485nm,发射=535nm),计算ROS含量。
结果显示,当使用不同浓度的Cu@Fe NPs处理A2780细胞时,细胞内ROS水平能够以剂量依赖性方式升高,且在72h观察点肿瘤细胞内ROS含量仍然较高(图4),这说明Cu@FeNPs通过向肿瘤细胞递送铜铁,诱发芬顿反应,使细胞处于持久且异常高的ROS内环境状态,最终诱发死亡。
Claims (3)
1.含铜铁氧体纳米粒子在制备抗肿瘤药物中的应用,所述的含铜铁氧体纳米粒子,其结构
式为Cu0.5Fe(II)0.5Fe(III)2O4,所述的抗肿瘤药物是抗三阴性乳腺癌或卵巢癌的药物。
2.根据权利要求1 所述的应用,其特征在于,是将CuCl2、FeCl2、FeCl3 溶于水中,使得Cu2+,Fe2+,Fe3+物质的量之比为1:1:4,加入聚乙烯吡咯酮,制得铜铁前驱体溶液,将铜铁前驱体溶液加热至85℃至90℃,然后滴加预热至85℃至90℃的含氢氧化钠和聚乙烯吡
咯酮的水溶液进行反应,反应后取出固体产物,依次用水、乙醇、丙酮和水洗涤,获得
洗涤后的产物,即为含铜铁氧体纳米粒子Cu0.5Fe(II)0.5Fe(III)2O4。
3.根据权利要求2 所述的应用,其特征在于,所述的铜铁前驱体溶液,其含0.1mM 的CuCl2,0.1mM 的FeCl2,0.4mM 的FeCl3 和5mg/ml 的聚乙烯吡咯酮,所述的含氢氧化钠和聚乙烯吡咯酮的水溶液是含1.6mM 的NaOH 和5mg/ml 的聚乙烯吡咯酮;将铜铁前驱体溶液加热至85℃至90℃,然后滴加预热至85℃至90℃的含氢氧化钠和聚乙烯吡咯酮的水溶液进行反应,反应后取出固体产物,依次用水、乙醇、丙酮和水洗涤,获得洗涤后的产
物。
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