CN111773245A - 一种复合纳米酶及其制备方法与应用 - Google Patents
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Abstract
本发明涉及一种白血病拮抗多肽修饰的Fe3O4‑Pt‑PEG复合纳米酶及其制备方法与应用。该复合纳米酶由Fe3O4纳米颗粒与Pt纳米颗粒通过聚乙二醇(PEG)修饰与连接而形成二元复合结构,Pt纳米颗粒一侧通过共价键链接趋化因子受体(CXCR4)拮抗多肽(E5)。通过高温热解法制备粒径分布均匀的磁性Fe3O4纳米颗粒,再利用末端带功能基团的PEG修饰Fe3O4纳米颗粒,然后利用颗粒表面功能基团吸附Pt离子并进行还原制备Pt纳米颗粒,最后用末端带功能基团的PEG进一步修饰Pt纳米颗粒并偶联E5。该制备方法简单方便、易于控制、成本低廉。制备的E5修饰复合纳米酶形貌尺寸均匀,具有良好的稳定性与生物相容性,能够催化氧化杀伤白血病细胞并抑制其转移,具有显著的治疗效果和潜在的应用价值。
Description
技术领域
本专利属于生物医学纳米技术领域,特别涉及一种白血病治疗的纳米药物及其制备方法与应用。
背景技术
Fe3O4纳米颗粒作为一种磁性纳米材料,是目前研究最多的金属氧化物纳米酶。Fe3O4纳米颗粒已经被广泛应用于生物医学、环境保护、疾病的诊断与治疗等领域,例如磁共振成像、药物递送、肿瘤检测与治疗等。2007年,阎课题组发现Fe3O4具有内在的类辣根过氧化物酶活性,能催化H2O2产生毒性的·OH,进一步氧化TMB或ABTS底物产生颜色变化。较小尺寸的Fe3O4纳米颗粒能够快速进入细胞,增加细胞内活性氧的产生并诱导细胞凋亡。
Pt纳米颗粒属于贵金属纳米酶,也被证明具有多种类酶活性,包括过氧化物酶、过氧化氢酶、氧化酶、超氧化物歧化酶。其酶催化活性受到反应pH、温度以及纳米颗粒的尺寸、浓度、表面修饰等因素的影响,例如:5nm Pt纳米颗粒类酶活性比20nm Pt颗粒高。Pt纳米颗粒能够代替天然酶,具有治疗活性氧介导的相关疾病的巨大潜力。
Fe3O4@Pt复合纳米酶结合了上述两种材料的优点,具有比表面积大、稳定性好、成本低、产量高、易于大规模工业生产,催化活性可控和可循环利用,具有产生大量活性氧的潜力,因此在肿瘤治疗方面具有广泛的应用前景。
小分子多肽E5是一种新型二聚体趋化因子受体4(CXCR4)拮抗多肽,是根据CXCR4的结构和序列特征设计的。E5可以动员骨髓中的白血病细胞转移到外周血,抑制白血病细胞向肝脏、脾脏的浸润。E5也可以阻断趋化因子受体4/趋化因子受体12(CXCR4/CXCL12)的相互作用并抑制白血病细胞的生长、粘附和浸润行为,从而有效提高白血病的治疗效果。
白血病患者经化疗后体内仍残留微量白血病细胞,为后续复发埋下了安全隐患,严重限制了白血病的临床治疗效果。近年来白血病的治疗有了很大的改善,完全缓解率明显升高,但患者预后仍不理想,转移和复发仍是临床治疗面临的主要挑战。目前尚无关于纳米酶治疗白血病的相关研究,因此有待深入探索功能化纳米酶在白血病治疗中的潜力。多肽E5对CXCR4/CXCL12轴有明显的抑制作用,纳米酶具有产生活性氧的能力,所以E5多肽修饰的Fe3O4@Pt纳米复合酶具有潜在的抗白血病疗效。
目前Fe3O4@Pt复合纳米酶的制备方法是直接高热解法合成油相Fe3O4@Pt复合纳米酶。但是此方法制备的复合纳米酶是油性的,生物相容性差,粒径分布不均,使得应用受到了很大的限制。另一种就是首先合成Fe3O4纳米颗粒,在Fe3O4纳米颗粒上直接诱导生成Pt纳米颗粒。此制备方法简便,生物相容性好,但是Pt纳米颗粒分布不均且容易脱落,稳定性和重复性不好。
发明内容
技术问题:针对上述情况,为克服现有技术之缺陷,本发明的目的是开发一种新颖的E5多肽修饰的Fe3O4-Pt纳米复合酶,通过结合纳米酶和CXCR4拮抗多肽阻断CXCR4/CXCL12信号轴,提高白血病的治疗效果。为了针对以往制备的复合纳米酶在应用过程中容易脱落和纳米颗粒分布不均,没有靶向性等问题,从而提供了一种制备复合纳米酶的方法,该方法操作简单、粒径均匀可控,易于控制、成本低廉。
技术方案:通过高温热解法制备粒径分布均匀的磁性Fe3O4纳米颗粒,再利用末端带功能基团的PEG修饰Fe3O4纳米颗粒,然后利用颗粒表面功能基团吸附Pt离子并进行还原制备Pt纳米颗粒,最后用末端带功能基团的PEG进一步修饰Pt纳米颗粒并偶联E5。具体包括以下内容:
本发明的复合纳米酶是一种白血病拮抗多肽修饰的Fe3O4-Pt-PEG复合纳米酶,该复合纳米酶由Fe3O4纳米颗粒与Pt纳米颗粒通过聚乙二醇PEG修饰与连接而形成二元复合结构,Pt纳米颗粒一侧通过共价键链接白血病细胞趋化因子受体CXCR4拮抗多肽E5。
本发明的一种白血病拮抗多肽修饰的Fe3O4-Pt-PEG复合纳米酶的制备方法如下:
步骤1,制备Fe3O4纳米颗粒:将乙酰丙酮铁和油酸按摩尔比为1:1~4:1分散于二苄醚中,控制铁浓度为3.4mg/mL,在恒定氮气2~10MPa气压下,通过控温程序装置以均匀加热速率3~5℃/min,从室温升温至200~250℃,并保持此温度50~70min,该过程中反应体系由砖红色逐渐变为黑色悬液;继续以相同的升温速率升温至280~320℃,并维持此温度30~40min,整个反应过程始终保持冷凝回流状态;反应结束冷却后,用无水乙醇洗涤3~5次并磁分离,充分去除油酸、二苄醚有机溶剂,通过外部磁体分离获得Fe3O4纳米颗粒,置于氯仿中保存备用,铁浓度为5mg/mL;
步骤2,制备聚乙二醇修饰的Fe3O4纳米颗粒即Fe3O4-PEG:将PEG、二环己基碳二亚胺、N-羟基琥珀酰亚胺、盐酸多巴胺、碳酸钠混合并溶解于二甲基甲酰胺、氯仿混合有机溶剂,并加入Fe3O4纳米颗粒,反应体系中各物质浓度依次为6mg/mL、0.6mg/mL、0.9mg/mL、0.4mg/mL、0.3mg/mL,35~40℃机械搅拌过夜,搅拌速度为500~1000r/min;反应结束后,将黑色产物倒入100mL烧杯中,加入正己烷后进行磁分离,弃上清,下部黑色物质经超声可分散于去离子水中,调节溶液pH值至7.4~7.7,透析去除多余的杂质,220nm的滤膜过滤去除较大尺寸的颗粒聚集体,得到Fe3O4-PEG纳米颗粒水溶液,铁浓度为5mg/mL,置于4℃保存备用;
步骤3,制备Fe3O4与Pt复合的纳米颗粒即Fe3O4-Pt-PEG纳米颗粒:Fe3O4-PEG纳米颗粒与Pt离子溶解于超纯水中,铁浓度为2mg/mL,铂离子与铁离子摩尔比为1:5~3:5,室温下搅拌5~12h,然后透析(选择截留分子量为3500~12000的透析袋)除去未吸附于Fe3O4@PEG表面的Pt离子,加入终浓度为0.2~0.6mg/mL还原剂还原Pt离子制备Pt纳米颗粒,并加入终浓度为0.1mg/mL的PEG进一步修饰Pt纳米颗粒,然后透析(选择截留分子量为3500~12000透析袋)除去游离的PEG,制得Fe3O4@Pt@PEG纳米颗粒水溶液,铁与铂浓度分别为1mg/mL、0.25mg/mL,于4℃冰箱保存备用;
步骤4,制备拮抗多肽E5修饰的Fe3O4@Pt@PEG纳米颗粒(Fe3O4@Pt@PEG@E5):将制备的Fe3O4@Pt@PEG纳米颗粒分散于含有EDC、NHS的MES缓冲液(pH=5.5,0.1mol/L),反应体系中铁、EDC、NHS浓度分别为1mg/mL、1.5mg/mL、1mg/mL,在室温下于摇床振荡(震荡速度为150rpm)反应2~5h,以活化Fe3O4@Pt@PEG表面的羧基。反应结束后,去离子水超滤离心3次除去游离杂质,超滤管截留分子量为30KDa,离心速度与时间为5000rpm与20min。取一定质量的E5多肽溶解于磷酸盐缓冲液中(pH=8,0.2mg/mL),然后加入上述洗涤后的纳米颗粒中,控制反应体系中铁浓度为1mg/mL,多肽与铁质量比为1:5~1:20,继续于摇床振荡反应12~24h,去离子水超滤离心3次除去游离多肽(超滤管MWCO=30KDa,4000rpm转速,30min),最终得到Fe3O4@Pt@E5纳米颗粒水溶液,铁与铂浓度为1mg/mL、0.25mg/mL。
此制备方法中所用PEG的分子量为2000、3500或者5000,分子式为COOH-PEG-SH或者COOH-PEG-NH2;所用的铂离子为氯铂酸钠、氯铂酸钾、次氯铂酸钠或次氯铂酸钾;所述的还原剂为硼氢化钠、柠檬酸钠或L-抗坏血酸。
所述的Fe3O4纳米颗粒的粒径为8~12nm,Pt纳米颗粒的粒径为1~2.5nm,其二元复合结构为PEG功能连接与修饰的1:1颗粒偶联体,形貌均匀,其水动力尺寸在20~35nm之间,表面电位为-28±4。
具有过氧化物酶、过氧化氢酶、氧化酶以及超氧化物歧化酶多酶活性。
超小的Pt纳米颗粒能够在肿瘤微环境中缓慢释放Pt离子。
能够靶向到白血病细胞并级联催化产生活性氧来有效杀伤白血病细胞,同时抑制白血病细胞浸润和转移。
有益效果:
1.本发明使用PEG修饰复合纳米酶,使颗粒具有良好的生物相容性和较长的血液循环时间,并且铁颗粒以化学键的形式与Pt颗粒结合,克服了颗粒之间结合不牢固、易脱落的缺点,为复合纳米酶更广泛应用奠定了基础。
2.本发明制备的E5多肽修饰的Fe3O4@Pt@PEG复合纳米酶具有良好的分散性、粒径小且分布均匀,稳定性好,催化活性高和循环应用,具有生成大量活性氧的潜力;制备方法操作简便、成本低廉和环保,产量高,易于大规模工业生产。
3.本制备方法通过结合纳米酶和CXCR4拮抗剂来提高细胞内活性氧水平并阻断CXCR4/CXCL12信号轴,并结合Pt离子杀伤作用,从而显著提高白血病的治疗效果,并防止白血病细胞转移或复发。
附图说明
图1为本发明白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶的制备过程示意图。
图2a为Fe3O4纳米颗粒的TEM图像;图2b为Fe3O4@Pt@PEG纳米颗粒的TEM图像;图2c为Fe3O4@Pt@PEG纳米颗粒的示意图和HRTEM图像;图2d为Fe3O4@Pt@PEG纳米颗粒的EDS能谱图。
图3a为DLS测量的Fe3O4@PEG和Fe3O4@Pt@PEG纳米颗粒的水动力尺寸;图3b为Fe3O4@Pt@PEG纳米颗粒的XRD图谱;图3c为Fe3O4@PEG和Fe3O4@Pt@PEG纳米颗粒的TGA图谱;图3d为Fe3O4@PEG和Fe3O4@Pt@PEG纳米颗粒的FTIR吸收光谱。
图4a为纳米颗粒过氧化物酶的活性研究;4b为纳米颗粒过氧化氢酶的活性研究;4c为纳米颗粒氧化酶的活性研究;4d为纳米颗粒超氧化物歧化酶的活性研究。
图5为Fe3O4@Pt@PEG纳米颗粒的铂离子累计释放曲线。
图6为本发明白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶的产生活性氧能力。
图7为本发明白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶的对细胞的毒性研究。
图8为本发明白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶的治疗效果图。
具体实施方式
本发明是一种白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶及其制备方法与应用,包括如下步骤:
解决的技术方案是,该制备方法是Fe3O4纳米颗粒与Pt纳米颗粒通过聚乙二醇(PEG)修饰与链接而形成二元复合结构,Pt纳米颗粒一侧通过共价键链接白血病细胞趋化因子受体(CXCR4)拮抗多肽(E5)(图1)。
该制备方法包括以下步骤:
(1)制备Fe3O4纳米颗粒:将7g乙酰丙酮铁、34mL油酸分散于100mL二苄基醚中,并倒入500mL三颈烧瓶中,在3MPa氮气气氛下,通过控温程序装置以3.2℃/min的均匀加热速率,从室温加热至230℃,并保持此温度60min,该过程中反应体系由砖红色逐渐变为黑色悬液。继续以相同的升温速率升温至280℃,并维持此温度20min,整个反应过程始终保持冷凝回流状态。反应结束冷却后,用无水乙醇超声(25KHz,2.5min,50%)洗涤4次,充分去除油酸、二苄醚有机溶剂,通过外部磁体分离获得Fe3O4纳米颗粒,置于氯仿中保存备用,铁浓度为5mg/mL;
(2)制备Fe3O4@PEG纳米颗粒:60mg COOH-PEG-SH(M=2000)、6mg二环己基碳二亚胺、9mg N-羟基琥珀酰亚胺、4mg盐酸多巴胺和3mg碳酸钠混合并溶解于3mL二甲基甲酰胺、7mL氯仿混合有机溶剂,加入10mg Fe3O4纳米颗粒,在35℃下机械搅拌(550r/min)过夜。反应结束后,将黑色产物倒入100mL烧杯中,加入正己烷后进行磁分离。弃上清,下部黑色物质经超声(30KHZ,3min,50%)可分散于去离子水中,调节溶液pH值至7.5。透析(透析袋MWCO=3500)去除多余的杂质,220nm的滤膜过滤去除较大尺寸的颗粒聚集体,得到Fe3O4@PEG纳米颗粒,铁浓度为5mg/mL,置于4℃保存备用;
(3)制备Fe3O4@Pt@PEG纳米颗粒:20mg Fe3O4@PEG纳米颗粒和5mg氯铂酸钾溶解于10mL的超纯水中,室温下搅拌(400rpm)8h,然后透析(透析袋MWCO=3500)去除未吸附于Fe3O4@PEG表面的Pt离子,加入0.4mg/mL的硼氢化钠还原Pt离子制备Pt纳米颗粒,并加入终浓度为0.1mg/mL的COOH-PEG-SH(M=2000)进一步修饰Pt纳米颗粒,制得Fe3O4@Pt@PEG纳米颗粒,铁与铂浓度分别为1mg/mL、0.25mg/mL,于4℃冰箱保存备用;
(4)制备Fe3O4@Pt@E5纳米颗粒:将制备的20mg Fe3O4@Pt@PEG纳米颗粒分散于20mLMES缓冲液中(pH=5.5,0.1mol/L,10mL),精密称取30mg EDC、20mg NHS溶解于该体系中,在室温下于摇床振荡(震荡速度为150rpm)反应5h,以活化Fe3O4@Pt@PEG表面的羧基。反应结束后,去离子水超滤(超滤管MWCO=30KDa,5000rpm转速,20min)离心3次除去反应中多余的EDC和NHS。取3mg E5多肽溶解于20mL磷酸盐缓冲液(pH=8,0.2mol/L),然后加入上述洗涤后的纳米颗粒中,继续于摇床振荡反应12h,去离子水超滤离心3次除去游离多肽(超滤管MWCO=30KDa,4000rpm转速,30min),最终得到Fe3O4@Pt@E5纳米颗粒,铁与铂浓度为1mg/mL、0.25mg/mL;
(5)所述的一种白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶的制备方法,其特征在于此制备方法中所用PEG的分子量为2000、3500或者5000,分子式为COOH-PEG-SH或者COOH-PEG-NH2;所用的铂离子为氯铂酸钠、氯铂酸钾、次氯铂酸钠或次氯铂酸钾;所述的还原剂为硼氢化钠、柠檬酸钠或L-抗坏血酸。
(6)所述的一种白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶,其特征在于所述的Fe3O4纳米颗粒的粒径为8~12nm,Pt纳米颗粒的粒径为1~2.5nm,其二元复合结构为PEG功能连接与修饰的1:1颗粒偶联体,形貌均匀,其水动力尺寸在20~35nm之间,表面电位为-28±4。
(7)所述的一种白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶,其特征在于具有过氧化物酶、过氧化氢酶、氧化酶以及超氧化物歧化酶多酶活性。
(8)根据权利要求1所述的一种白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶,其特征在于超小的Pt纳米颗粒能够在肿瘤微环境中缓慢释放Pt离子。
(9)根据权利要求1所述一种白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶,其特征在于,能够靶向到白血病细胞并级联催化产生活性氧来有效杀伤白血病细胞,同时抑制白血病细胞浸润和转移。
为了更好地理解本发明,下面用实施例进一步阐明本发明,但本发明的内容不仅仅局限于下面实例
实施例1
Fe3O4纳米颗粒的制备
将3.5g乙酰丙酮铁、15mL油酸分散于50mL二苄基醚中,并倒入250mL三颈烧瓶中,在4MPa氮气气氛下,通过控温程序装置以3.2℃/min的均匀加热速率,从室温加热至220℃,并保持此温度50min,该过程中反应体系由砖红色逐渐变为黑色悬液。继续以相同的升温速率升温至290℃,并维持此温度30min,整个反应过程始终保持冷凝回流状态。反应结束冷却后,用无水乙醇超声(30KHz,2min,60%)洗涤5次,充分去除油酸、二苄醚有机溶剂,通过外部磁体分离获得Fe3O4纳米颗粒,置于氯仿中保存备用,铁浓度为5mg/mL。
实施例2
Fe3O4@PEG纳米颗粒的制备
120mg COOH-PEG-NH2(M=5000)、12mg二环己基碳二亚胺、18mg N-羟基琥珀酰亚胺、8mg盐酸多巴胺和6mg碳酸钠混合并溶解于3mL二甲基甲酰胺、7mL氯仿混合有机溶剂,加入20mg Fe3O4纳米颗粒,在35℃下机械搅拌(600r/min)过夜。反应结束后,将黑色产物倒入100mL烧杯中,加入正己烷后进行磁分离。弃上清,下部黑色物质经超声(20KHZ,4min,40%)可分散于去离子水中,调节溶液pH值至7.6。透析(透析袋MWCO=8000)去除多余的杂质,220nm的滤膜过滤去除较大尺寸的颗粒聚集体,得到Fe3O4@PEG纳米颗粒,铁浓度为5mg/mL,置于4℃保存备用。
实施例3
Fe3O4@Pt@PEG纳米颗粒的制备
10mg Fe3O4@PEG纳米颗粒和2.5mg次氯铂酸钾溶解于10mL的超纯水中,室温下搅拌(500rpm)12h,然后透析(透析袋MWCO=8000)去除未吸附于Fe3O4@PEG表面的Pt离子,加入0.5mg/mL的柠檬酸钠还原Pt离子制备Pt纳米颗粒,并加入终浓度为0.1mg/mL的COOH-PEG-NH2(M=5000)进一步修饰Pt纳米颗粒,制得Fe3O4@Pt@PEG纳米颗粒,铁与铂浓度分别为1mg/mL、0.25mg/mL,于4℃冰箱保存备用。
取少量上述步骤制得的Fe3O4@PEG和Fe3O4@Pt@PEG纳米颗粒,涂在铜网上,利用透射电镜观察颗粒形貌和粒径。图2a所示,可以观察到Fe3O4纳米颗粒呈球状,粒径分布均匀(8~12nm),无团聚现象,稳定性好;图2b可以明显的看到具有Janus结构的Fe3O4@Pt@PEG纳米颗粒,Pt纳米颗粒的粒径大约是2nm左右;图2c可以清晰的看到Fe3O4和Pt颗粒的晶格条纹,进一步证实了Fe3O4和Pt颗粒的存在;我们分析了Fe3O4@Pt@PEG纳米颗粒的元素组成和比例,图2d EDS能谱结果表明其主要含有Fe、O、C、N、S、Pt元素,Fe与Pt原子比例约为3:1,所制备的复合纳米酶中含有预期的基本元素。
实施例4
Fe3O4@Pt@E5纳米颗粒的制备
将制备的10mg Fe3O4@Pt@PEG纳米颗粒分散于10mL MES缓冲液中(pH=5.5,0.1mol/L,10mL),精密称取15mg EDC、10mg NHS溶解于该体系中,在室温下于摇床振荡(震荡速度为150rpm)反应3h,以活化Fe3O4@Pt@PEG表面的羧基。反应结束后,去离子水超滤(超滤管MWCO=30KDa,5000rpm转速,20min)离心3次除去反应中多余的EDC和NHS。取1.5mg E5多肽溶解于10mL磷酸盐缓冲液(pH=8,0.2mol/L),然后加入上述洗涤后的纳米颗粒中,继续于摇床振荡反应24h,去离子水超滤离心3次除去游离多肽(超滤管MWCO=30KDa,4000rpm转速,30min),最终得到Fe3O4@Pt@E5纳米颗粒,铁与铂浓度为1mg/mL、0.25mg/mL。
如图3a所示,Fe3O4@Pt@PEG纳米颗粒(30±3nm)的水动力尺寸大于Fe3O4@PEG纳米颗粒(25±2nm)。XRD图谱(图3b)所示,通过与标准JCPDS卡片对照,2θ为21.2°、35.1°、41.4°、50.4°、62.9°、67.2°、67.3°的衍射峰完全符合Fe3O4立方晶体结构(111)、(220)、(311)、(400)、(422)、(511)、(440)。也观察到2θ在27.9、38.3和48.1衍射峰的存在,与Pt晶体的(001)、(110)、(111)相匹配。纳米颗粒的热重分析曲线如图3c所示,在0~100℃发生首次失重,是纳米颗粒表面的水分子损失。样品在300℃~410℃温度范围内再次发生失重,Fe3O4@PEG和Fe3O4@Pt@PEG纳米颗粒分别损失50%、64%重量,可以归因于纳米颗粒表面PEG分子被将解。Fe3O4@PEG和Fe3O4@Pt@PEG纳米颗粒的红外图谱(图3d)显示了分别在1080、1670和3440cm-1处发现了与-SH、C=O、-OH基团相对应的特征峰,这证实了纳米颗粒表面成功修饰了PEG。
实施例5
Fe3O4@Pt@PEG复合纳米酶的酶活性研究
纳米颗粒带负电荷,TMB带正电荷。两者相互吸引并具有很强的亲和力。因此在H2O2存在下,通过催化TMB底物,在653nm波长处测量形成的蓝色产物的吸光度来研究过氧化物酶的活性。研究了时间(0~20min)、温度(20~80℃)、pH(2~11)、纳米颗浓度(铁的浓度0.05~0.5μg/mL)的依赖性。此外,利用溶解氧电极检测氧气的生成速率以验证过氧化氢酶的活性。在20mL不同pH(2~11)的缓冲液中加入不同量的(铁的浓度0.0~10μg/mL)纳米颗粒和3mL 30%H2O2,然后插入溶解氧电极,设置相关参数,对反应体系中的氧溶解度进行30min实时监测。氧化酶活性研究是通过氧化不同浓度(1~4mmol/L)的葡萄糖产生H2O2,产生的H2O2进一步氧化TMB变色并测定其吸光度。O2·-能将氮蓝四唑还原生成蓝色甲瓒,超氧化物歧化酶可以清除O2·-并抑制蓝色产物的形成。因此,在560nm波长处,测定了不同浓度纳米颗粒(Fe浓度20~200μg/mL)在黄嘌呤/黄嘌呤氧化酶体系中对蓝色甲瓒的抑制率和对O2·-的清除效率,以研究纳米颗粒的超氧化物歧化酶活性。
如图4所示,Fe3O4@Pt@PEG复合纳米酶展现出与天然酶相似的过氧化物酶、过氧化氢酶、氧化酶、超氧化物歧化酶催化效率。纳米颗粒对TMB的催化作用具有时间和浓度依赖性,随着纳米颗粒用量的增加,催化活性显著增强。纳米颗粒在较宽的温度范围(20~80℃)内具有较强的酶活性,pH=4.0时酶活性最高。纳米颗粒的类过氧化物酶活性顺序为Fe3O4@Pt@PEG>Pt@PEG>Fe3O4@PEG(图4a)。如图4b所示,纳米颗粒的过氧化氢酶活性与浓度、时间密切相关,最优pH值是碱性。与Fe3O4@PEG和Pt@PEG相比,Fe3O4@Pt@PEG具有最高的过氧化氢酶催化活性。Fe3O4@Pt@PEG也可以氧化葡萄糖生成H2O2,具有氧化酶活性,纳米颗粒浓度、葡萄糖浓度与反应体系的吸光度呈良好的线性关系(图4c)。Fe3O4@Pt@PEG纳米颗粒也具有较强的超氧化物歧化酶活性,其酶活性对时间和纳米颗粒浓度具有较强的依赖性(图4d)。
实施例6
Fe3O4@Pt@PEG复合纳米酶的铂离子累积释放
将30mg Fe3O4@Pt@E5纳米颗粒分散于20mL PBS中,再转移至透析袋中(MWCO=3500),将透析袋置于500mL PBS透析液中,在37℃的摇床震荡(100rpm)4天。在规定的时间间隔从透析液中取出1mL样本。采样后,立即用等量的新鲜PBS补充透析液,通过ICP-MS测量铂离子的累积释放量。
如图5所示,纳米颗粒在96小时累积释放出30%以上的铂离子。铂离子可以作为化疗药物杀死癌细胞,联合使用铂纳米颗粒与释放的铂离子协同治疗白血病可提高治疗效果。
实施例7
纳米颗粒在细胞内产生活性氧能力
以DCFH-DA为荧光探针检测细胞内活性氧水平,验证纳米颗粒在HL-60细胞内产生活性氧的能力。HL-60细胞(5×106个/孔)分别与纳米颗粒和N-乙酰半胱氨酸(NAC)共孵育12h,已知NAC可以减少细胞内活性氧。将准备的DCFH-DA染液通过离心处理代替细胞培养液,并与细胞再孵育30min。通过流式细胞术检测HL-60细胞内活性中间体产生的荧光强度。
如图6所示,与对照组相比,Fe3O4@Pt@PEG复合纳米酶处理的HL-60细胞内有强烈的荧光信号,表明Fe3O4@Pt@PEG能在细胞内产生过量的活性氧。其中,未加NAC组的荧光强度明显强于加NAC组。
实施例8
白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶体外细胞毒性测定
体外细胞毒性实验是采用CCK8试剂盒测量了纳米颗粒对HL-60细胞活性的影响。将密度为1×105个/孔的HL-60细胞与不同浓度纳米颗粒(Fe浓度为50,100,200μg/mL,Pt浓度为12.5,25,50μg/mL)在96孔板中孵育12h、24h、48h。孵育结束后,每孔加入10μL CCK-8,于恒温培养箱中继续孵育4h,然后在酶标仪上于450nm处检测吸光度。进行6次独立实验,计算细胞平均存活率(%)。
如图7所示,随着纳米颗粒浓度的增加与培养时间的延长,细胞活力明显下降,Fe3O4@Pt@E5纳米颗粒能显著地降低细胞活性。
实施例9
建立白血病小鼠模型
NOD/SCID小鼠(16-18g)在实验前3天适应实验室环境。用250cGy剂量的X射线照射小鼠,第二天尾静脉注射100μL HL-60细胞悬液(1.2×106个)。骨髓为细胞的增殖提供了重要的环境,因此注射的HL-60细胞首先快速迁移至骨髓,然后浸润至脾脏和外周血。因此,在HL-60细胞接种的第20、30、40天,收集小鼠的骨髓、脾脏和外周血制备单细胞悬液。红细胞裂解后,通过流式细胞仪分析HL-60细胞(CD33标记)的比例。收集新鲜的外周血和骨髓积液,立即涂片并用瑞氏-吉姆萨染料染色,以观察骨髓和外周血中是否存在HL-60细胞。
结果显示,随着时间延长HL-60细胞在骨髓中占总细胞的6.1%、40%、91.9%,在脾脏中占3.3%、10.2%、65.2%,在血液中占0.71%、3.32%、54.7%,说明白血病小鼠模型构建成功。在显微镜下可以直接观察到外周血和骨髓腔积液图片上有细胞核大、深蓝紫色的HL-60细胞存在,进一步验证了成功构建了动物模型。
实施例10
白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶对白血病小鼠的治疗效果
白血病小鼠模型构建成功后,小鼠被随机分为六组(n=7)。分别尾静脉注射PBS、E5、Fe3O4@PEG、Pt@PEG、Fe3O4@Pt@PEG、Fe3O4@Pt@E5,注射剂量铁、铂的浓度是10mg/kg、2.5mg/kg,每周治疗两次,共治疗3周,每天检测小鼠的体重。治疗21天后,收集骨髓、脾脏和外周血细胞,监测骨髓、脾脏和外周血细胞中HL-60细胞的变化来评价治疗效果。
如图8所示,与对照组相比,所有纳米颗粒组的骨髓、脾脏和外周血中HL-60细胞比例均下降,其中Fe3O4@Pt@E5组降幅最大。Fe3O4@Pt@E5组骨髓中的HL-60细胞比例由92.1%降至14.5%,脾脏中的HL-60细胞比例由53.1%降至2.21%,外周血中的HL-60细胞比例由54.2%降至1.12%。
实施例11
白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶的药代动力学、生物分布和代谢研究
为了研究Fe3O4@Pt@E5的药代动力学,尾静脉注射Fe3O4@Pt@E5后,采集10μL血液样本于含有肝素钠的生理盐水中,使用ICP-MS测量了血液中铂离子浓度随时间(2min、5min、10min、20min、30min、1h、2h、4h、8h和24h)的变化。计算了Fe3O4@Pt@E5的血液半衰期。治疗结束后,称取一些解剖的内脏组织并用王水消解,用ICP-MS定量测定内脏中铂离子含量。其他组织切片用普鲁士蓝染色,以观察治疗后纳米颗粒在器官组织中的分布。
在药代动力学实验中得到了Fe3O4@Pt@E5的血液清除曲线,计算出血液半衰期为12.5h。普鲁士蓝染色结果显示,纳米颗粒主要聚集在肝脏和脾脏,也有少量分布在肺和肾中。ICP-MS结果与普鲁士蓝染色结果一致,纳米颗粒主要通过肝脏和脾脏代谢。
实施例12
白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶的体内毒性研究
为了评价Fe3O4@Pt@E5的生物系统毒性,BALB/c小鼠被分成PBS组和Fe3O4@Pt@E5组,每周治疗两次,连续治疗3周。在治疗期间,每天监测所有小鼠的体重。治疗结束后收集血样和内脏组织(心、肝、脾、肺、肾)进行进一步毒性研究。对血样进行相应处理后,检测肝功能、肾功能生化指标(丙氨酸氨基转移酶、天冬氨酸氨基转移酶、肌酐、尿素)。内脏组织被切片和H&E染色,以方便观察病变。
从组织的H&E染色中可以观察到脏器组织结构完整,没有炎症细胞浸润,未见明显异常病理改变。Fe3O4@Pt@E5组与对照组相比体重无异。肝功能、肾功能生化指标结果显示,除了Fe3O4@Pt@E5组的ALT指标稍微下降外,其余指标与对照组相比均无显著性差异,说明Fe3O4@Pt@E5组小鼠肝、肾功能正常,无损伤或病变。以上结果证明Fe3O4@Pt@E5具有良好的安全性,对小鼠机体无显著影响。
Claims (7)
1.一种白血病拮抗多肽修饰的Fe3O4-Pt-PEG复合纳米酶,其特征在于,该复合纳米酶由Fe3O4纳米颗粒与Pt纳米颗粒通过聚乙二醇PEG修饰与连接而形成二元复合结构,Pt纳米颗粒一侧通过共价键链接白血病细胞趋化因子受体CXCR4拮抗多肽E5。
2.一种如权利要求1所述一种白血病拮抗多肽修饰的Fe3O4-Pt-PEG复合纳米酶的制备方法,其特征在于,其制备方法如下:
步骤1,制备Fe3O4纳米颗粒:将乙酰丙酮铁和油酸按摩尔比为1:1~4:1分散于二苄醚中,控制铁浓度为3.4mg/mL,在恒定氮气2~10MPa气压下,通过控温程序装置以均匀加热速率3~5℃/min,从室温升温至200~250℃,并保持此温度50~70min,该过程中反应体系由砖红色逐渐变为黑色悬液;继续以相同的升温速率升温至280~320℃,并维持此温度30~40min,整个反应过程始终保持冷凝回流状态;反应结束冷却后,用无水乙醇洗涤3~5次并磁分离,充分去除油酸、二苄醚有机溶剂,通过外部磁体分离获得Fe3O4纳米颗粒,置于氯仿中保存备用,铁浓度为5mg/mL;
步骤2,制备聚乙二醇修饰的Fe3O4纳米颗粒即Fe3O4-PEG:将PEG、二环己基碳二亚胺、N-羟基琥珀酰亚胺、盐酸多巴胺、碳酸钠混合并溶解于二甲基甲酰胺、氯仿混合有机溶剂,并加入Fe3O4纳米颗粒,反应体系中各物质浓度依次为6mg/mL、0.6mg/mL、0.9mg/mL、0.4mg/mL、0.3mg/mL,35~40℃机械搅拌过夜,搅拌速度为500~1000r/min;反应结束后,将黑色产物倒入100mL烧杯中,加入正己烷后进行磁分离,弃上清,下部黑色物质经超声可分散于去离子水中,调节溶液pH值至7.4~7.7,透析去除多余的杂质,220nm的滤膜过滤去除较大尺寸的颗粒聚集体,得到Fe3O4-PEG纳米颗粒水溶液,铁浓度为5mg/mL,置于4℃保存备用;
步骤3,制备Fe3O4与Pt复合的纳米颗粒即Fe3O4-Pt-PEG纳米颗粒:Fe3O4-PEG纳米颗粒与Pt离子溶解于超纯水中,铁浓度为2mg/mL,铂离子与铁离子摩尔比为1:5~3:5,室温下搅拌5~12h,然后透析(选择截留分子量为3500~12000的透析袋)除去未吸附于Fe3O4@PEG表面的Pt离子,加入终浓度为0.2~0.6mg/mL还原剂还原Pt离子制备Pt纳米颗粒,并加入终浓度为0.1mg/mL的PEG进一步修饰Pt纳米颗粒,然后透析(选择截留分子量为3500~12000透析袋)除去游离的PEG,制得Fe3O4@Pt@PEG纳米颗粒水溶液,铁与铂浓度分别为1mg/mL、0.25mg/mL,于4℃冰箱保存备用;
步骤4,制备拮抗多肽E5修饰的Fe3O4@Pt@PEG纳米颗粒(Fe3O4@Pt@PEG@E5):将制备的Fe3O4@Pt@PEG纳米颗粒分散于含有EDC、NHS的MES缓冲液(pH=5.5,0.1mol/L),反应体系中铁、EDC、NHS浓度分别为1mg/mL、1.5mg/mL、1mg/mL,在室温下于摇床振荡(震荡速度为150rpm)反应2~5h,以活化Fe3O4@Pt@PEG表面的羧基。反应结束后,去离子水超滤离心3次除去游离杂质,超滤管截留分子量为30KDa,离心速度与时间为5000rpm与20min。取一定质量的E5多肽溶解于磷酸盐缓冲液中(pH=8,0.2mg/mL),然后加入上述洗涤后的纳米颗粒中,控制反应体系中铁浓度为1mg/mL,多肽与铁质量比为1:5~1:20,继续于摇床振荡反应12~24h,去离子水超滤离心3次除去游离多肽(超滤管MWCO=30KDa,4000rpm转速,30min),最终得到Fe3O4@Pt@E5纳米颗粒水溶液,铁与铂浓度为1mg/mL、0.25mg/mL。
3.根据权利要求2所述的一种白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶的制备方法,其特征在于此制备方法中所用PEG的分子量为2000、3500或者5000,分子式为COOH-PEG-SH或者COOH-PEG-NH2;所用的铂离子为氯铂酸钠、氯铂酸钾、次氯铂酸钠或次氯铂酸钾;所述的还原剂为硼氢化钠、柠檬酸钠或L-抗坏血酸。
4.根据权利要求1所述的一种白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶,其特征在于所述的Fe3O4纳米颗粒的粒径为8~12nm,Pt纳米颗粒的粒径为1~2.5nm,其二元复合结构为PEG功能连接与修饰的1:1颗粒偶联体,形貌均匀,其水动力尺寸在20~35nm之间,表面电位为-28±4。
5.根据权利要求1所述的一种白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶,其特征在于具有过氧化物酶、过氧化氢酶、氧化酶以及超氧化物歧化酶多酶活性。
6.根据权利要求1所述的一种白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶,其特征在于超小的Pt纳米颗粒能够在肿瘤微环境中缓慢释放Pt离子。
7.根据权利要求1所述一种白血病拮抗多肽修饰的Fe3O4@Pt@PEG复合纳米酶,其特征在于,能够靶向到白血病细胞并级联催化产生活性氧来有效杀伤白血病细胞,同时抑制白血病细胞浸润和转移。
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