CN117045820A - 一种兼具大孔和微孔尺寸的核壳结构金属有机框架复合材料制备及其应用 - Google Patents
一种兼具大孔和微孔尺寸的核壳结构金属有机框架复合材料制备及其应用 Download PDFInfo
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Abstract
本发明公开了一种兼具大孔和微孔尺寸的核壳结构金属有机框架复合材料制备及其应用,属于生物医学和材料化学领域,本发明采用酸刻蚀策略合成负载大分子葡萄糖氧化酶的中空介孔普鲁士蓝金属有机框架材料,异质结外延生长法在中空介孔普鲁士蓝表面生长壳层ZIF‑8,壳层的ZIF‑8既可以有效防止葡萄糖氧化酶的泄露又可为小分子5‑氟尿嘧啶药物提供装载空间,可实现同时装载两种抗癌药物的核壳结构金属有机框架复合材料。本发明合成的复合材料尺寸均匀,实现MOF‑on‑MOF的孔道分级策略,实现对大小客体分子的有效封载,扩展了实际应用领域利用两种抗癌药物的联合作用,进而实现治疗癌症的需求,具有一定的临床应用潜能。
Description
技术领域
本发明涉及一种兼具大孔和微孔尺寸的核壳结构金属有机框架复合材料制备及其应用,属于生物医学和材料化学领域。
背景技术
癌症严重威胁人类的生命健康,影响着人类生活质量,也成为全球性的重大公共健康难题,并且其发病率还在不断增加。近年来,葡萄糖氧化酶在肿瘤治疗中得到了广泛的应用。葡萄糖氧化酶催化葡萄糖氧化成葡萄糖酸和有毒的过氧化氢,饿死和杀死肿瘤细胞,即所谓的饥饿疗法,过氧化氢可以进一步转化为高毒性的·OH用于化学动力治疗。饥饿治疗与化学动力治疗相结合,达到超加性抗肿瘤效果。5-氟尿嘧啶是一种胸苷酸合成酶抑制药,在细胞内转变为5-氟尿嘧啶脱氧核苷酸(5F-dUMP),而抑制脱氧胸苷酸合成酶,阻止脱氧尿苷酸(dUMP)甲基化转变为脱氧胸苷酸(dTMP),从而影响DNA的合成。研究表明饥饿治疗协同化疗具有非常有效的抗肿瘤的作用。
金属有机框架材料(Metal-Organic Framework,MOFs)是由有机配体与金属离子经过自组装形成的一维、二维或者三维的周期性多孔材料,具有多孔性,极大的比表面积、可调节的孔径和可调节的孔隙、易于功能化、催化活性等优势,这些显著的优势使得金属有机框架材料在肿瘤治疗方面显示出巨大的应用潜力。其中,介孔金属有机框架凭借较大的孔内径可以作为大分子蛋白药物优越的载体,但单一的介孔金属有机框架在药物递送过程中发生提前泄露的问题尚未得到有效解决。
发明内容
针对上述现有技术中的不足,本发明提供一种兼具大孔和微孔尺寸的核壳结构金属有机框架复合材料制备及其应用,所述制备方法制备过程简单,易于操作,流程稳定可靠。所述HMPB@ZIF-8同时负载葡萄糖氧化酶和5-氟尿嘧啶两种抗癌药物。本发明通过对介孔金属有机框架进行孔道分级,装载大分子葡萄糖氧化酶蛋白及小分子化疗药物5-氟尿嘧啶,实现了联合肿瘤治疗。
为实现上述目的,本发明采用如下技术方案:
一种兼具大孔和微孔尺寸的核壳结构金属有机框架复合材料,负载的抗癌药物为葡萄糖氧化酶和5-氟尿嘧啶,所述框架复合材料的分子式为HMPB@GOx@ZIF-8@5-Fu@HA,其中复合材料中葡萄糖氧化酶封装在介孔HMPB内,所述5-氟尿嘧啶吸附进入ZIF-8的壳层内。
进一步地,上述核壳结构金属有机框架复合材料中,所述葡萄糖氧化酶载药量可以达到15.51wt%,5-氟尿嘧啶载药量可以达到19.33wt%;所述HMPB以聚乙烯吡咯烷为配体,铁离子为金属离子,ZIF-8以2-甲基咪唑为配体,锌离子为金属离子,利用水热法合成的纳米尺寸的核壳结构金属有机骨架材料。
上述核壳结构金属有机框架材料的制备方法,包括以下步骤:溶剂热法合成介孔HMPB,将葡萄糖氧化酶封装在介孔空腔内部,外延生长壳层ZIF-8作为控释开关,接着5-氟尿嘧啶吸附进入ZIF-8的壳层中,以透明质酸作为靶向剂将药物靶向递送到肿瘤组织,最终得到负载葡萄糖氧化酶和5-氟尿嘧啶的核壳结构金属有机框架复合材料,也可称之为HMPB@GOx@ZIF-8@5-Fu@HA。
进一步的,所述HMPB是一种金属有机框架(MOF)材料,HMPB可以通过现有技术中公开的方法进行制备。
进一步的,合成负载葡萄糖氧化酶的金属有机框架复合材料(HMPB@GOx)时,在常温下搅拌反应,搅拌转速为500r/min,搅拌时间为12小时。
进一步的,合成核壳结构金属有机框架复合材料(HMPB@GOx@ZIF-8)时,聚(4-苯乙烯磺酸钠)改性液浓度为0.3wt%。
进一步的,合成负载5-氟尿嘧啶的核壳结构金属有机框架复合材料(HMPB@GOx@ZIF-8@5-Fu@HA)时,5-氟尿嘧啶与透明质酸浓度分别为2mg/mL和1.5mg/mL。
按照上述方法得到的负载葡萄糖氧化酶的金属有机框架复合材料(HMPB@GOx),外延生长ZIF-8,壳层ZIF-8可以继续物理吸附法吸附5-氟尿嘧啶,最终得到同时负载葡萄糖氧化酶和5-氟尿嘧啶的核壳结构金属有机框架复合材料(HMPB@GOx@ZIF-8@5-Fu@HA)。
本发明通过ZIF-8来划分介孔的孔隙空间,小分子5-FU通过物理吸附的方式占据壳层ZIF-8的孔隙空间的方式,使其具备pH响应及葡萄糖相应释放能力,相当程度上满足了两种抗癌药物协同治疗肿瘤作用,可进一步增强抗肿瘤疗效和减少耐药性的产生,具有很好的临床应用前景。
本发明基于水热法,采用先“溶剂热法”合成负载葡萄糖氧化酶的金属有机框架复合材料(HMPB@GOx),再利用MOF-On-MOF壳层材料的多孔吸附性,将小分子5-氟尿嘧啶物理吸附进入ZIF-8的空隙中。本发明合成的负载两种抗癌药物的核壳结构金属有机框架复合材料相比于负载一种抗癌药物的金属有机框架复合材料,更加充分利用MOF材料的孔隙,并且显示出更好的抗肿瘤效果并减少药物提前释放的产生。除此,经过实验证实,该材料在两种药物释放过程中均呈现良好的pH控制释放效果,极大地推进多种不同类型的抗癌药物协同作用在癌症组织治疗领域的应用进展。
本发明具体的有益效果在于:
(1)本发明采用先“溶剂热法”合成介孔材料、后物理吸附的策略合成的负载两种抗癌药物的核壳结构金属有机框架复合材料,制备方法简单,流程稳定可靠。通过外延生长ZIF-8作为壳层结构,实现了两个大小客体分子的有效封装,不仅极大地增强了介孔金属有机框材料的孔道利用率,还提高了总体的载药量。经验证,葡萄糖氧化酶载药量可以达到15.51wt%,5-氟尿嘧啶载药量可以达到19.33wt%。
(2)本发明负载两种抗癌药物的核壳结构金属有机框架复合材料在药物释放过程中均呈现良好的控制释放效果。大分子葡萄糖氧化酶封在介孔中,而小分子5-氟尿嘧啶吸附进入ZIF-8划分的壳层中,由于空间限制效应引起了较强的主客体相互作用,可避免葡糖糖氧化酶的提前释放。
(3)本发明利用核壳结构金属有机框架材料装载两种不同类型的抗癌药物:葡萄糖氧化酶和5-氟尿嘧啶,起到全面的、两者协同的抗癌作用,提高葡萄糖氧化酶的抗癌活性,降低5-氟尿嘧啶的毒副作用,能达到抗癌作用的长效性、缓释性的特点,为抗肿瘤方面的药物应用提供新方向,为其临床应用提供了潜在的可能性。
附图说明
图1为HMPB@ZIF-8的X射线衍射(XRD)图;
图2为透射电镜(TEM)图和SEM元素映射图像,其中,a为HMPB@GOx@ZIF-8@5-Fu的透射电镜图,b,c为HMPB的透射电镜图,d,e,f为HMPB@GOx@ZIF-8@5-Fu单颗的透射电镜图,h为HMPB@GOx@ZIF-8@5-Fu的元素映射图;
图3为HMPB、HMPB@GOx、HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu的XPS能谱图;
图4为GOx和5-Fu标准溶液的紫外吸收曲线图和装载GOx和5-Fu前后的上清液紫外吸收曲线变化图;
图5为药物释放曲线图,其中,a为HMPB@GOx@ZIF-8@5-Fu@HA在不同pH缓冲溶液中的GOx释放曲线,b为HMPB@GOx@ZIF-8@5-Fu@HA在不同pH缓冲溶液中的5-Fu释放曲线,c为HMPB@GOx@ZIF-8@5-Fu@HA在不同pH缓冲溶液中的Zn2+释放曲线,DHMPB@GOx@ZIF-8@5-Fu@HA在不同pH葡萄糖溶液中的Zn2+释放曲线;
图6为HMPB@GOx@ZIF-8@5-Fu@HA在葡萄糖溶液中的酶活性图,其中a为等量的酶、HMPB@GOx、HMPB@GOx@ZIF-8@5-Fu@HA在葡萄糖溶液中pH变化曲线,b为不同浓度的HMPB@GOx@ZIF-8@5-Fu@HA在葡萄糖溶液中葡萄糖消耗曲线,c为HMPB@GOx@ZIF-8@5-Fu@HA在pH=7.4的葡萄糖溶液或pH=7.4的缓冲溶液中EPR波谱,图d为不同浓度下的HMPB@GOx@ZIF-8@5-Fu@HA与TMB反应曲线,图e为不同浓度下的HMPB@GOx@ZIF-8@5-Fu@HA与MB反应曲线;
图7为材料对细胞的毒性,其中图a为不同浓度下HMPB、HMPB@GOx、HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu、HMPB@GOx@ZIF-8@5-Fu@HA对4T1细胞毒性图,其中图b为不同浓度下HMPB、HMPB@GOx、HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu、HMPB@GOx@ZIF-8@5-Fu@HA对3T3细胞毒性图;
图8为HMPB@GOx@ZIF-8@5-Fu@HA与4T1细胞共孵育不同时间的细胞摄取图;
图9为HMPB、HMPB@GOx、HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu、HMPB@GOx@ZIF-8@5-Fu@HA与4T1细胞共孵育6小时的细胞活死染色图;
图10为HMPB、HMPB@GOx、HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu、HMPB@GOx@ZIF-8@5-Fu@HA与4T1细胞共孵育6小时细胞内的ROS生成图;
图11为HMPB、HMPB@GOx、HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu、HMPB@GOx@ZIF-8@5-Fu@HA与4T1细胞共孵育6小时细胞内的线粒体膜电位变化JC-1染色图;
图12为HMPB、HMPB@GOx、HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu、HMPB@GOx@ZIF-8@5-Fu@HA与4T1细胞共孵育6小时流式细胞凋亡图。
具体实施方式
下面将结合实例进一步阐述本发明的内容,但这些实例并不限制本发明的保护范围,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。
实施例1
(1)采用溶剂热法,将3.8g的PVP和110mg的K3Fe(CN)6添加于50mL的0.1M HCl,超声30min使其充分分散,放置于80℃烘箱24h,溶液由透明黄色变成深蓝色浑浊液,14000rpm/min 15min离心分离PB NPs,蒸馏水洗涤三次乙醇洗涤一次,50℃过夜烘干得到PB NPs,之后将20mg的PB NPs与100mg PVP超声混合分散于1M 20mL的HCl中,高压反应釜140℃刻蚀2h,最后10000rpm/min 12min蒸馏水离心洗涤三次,乙醇洗涤一次,50℃过夜烘干得到5.45mg立方体普鲁士蓝纳米颗粒,简称HMPB。
(2)将10mg的葡萄糖氧化酶和上述制备的10mg HMPB超声均匀溶解于10mL的超纯水中,在常温的条件下以600r/min的转速搅拌反应12小时,收集离心并用去离子水洗涤3次,冷冻干燥得到10.6mg负载葡萄糖氧化酶的介孔普鲁士蓝材料,简称HMPB@GOx。
(3)将10mg上述HMPB@GOx与120mg聚(4-苯乙烯磺酸钠)均匀混合在40mL超纯水中,在常温的条件下以600r/min的转速搅拌反应0.5h,10000rpm/min 12min离心分离并再次超声分散在15mL超纯水中,向其中加入1mL 0.0672M的六水合硝酸锌,在常温的条件下以600r/min的转速搅拌反应0.5h,随后再向溶液中加入2mL0.1096 M的2-甲基咪唑,在常温的条件下以600r/min的转速搅拌反应0.5h,收集离心并用去离子水洗涤3次,冷冻干燥得到10.3mg ZIF-8作为壳层并负载葡萄糖氧化酶的介孔普鲁士蓝材料,简称HMPB@GOx@ZIF-8。
(4)将10mg上述HMPB@GOx@ZIF-8与20mg 5-氟尿嘧啶均匀混合在10mL超纯水中,并向其中投入8mg葡萄糖氧化酶在常温的条件下以600r/min的转速搅拌反应12h,收集离心并用去离子水洗涤3次,冷冻干燥得到10.1mg ZIF-8作为壳层并负载葡萄糖氧化酶和5-氟尿嘧啶的介孔普鲁士蓝材料,简称HMPB@GOx@ZIF-8@5-Fu。
(5)将10mg上述HMPB@GOx@ZIF-8与20mg 5-氟尿嘧啶以及15mg透明质酸均匀混合在10mL超纯水中,并向其中投入8mg葡萄糖氧化酶在常温的条件下以600r/min的转速搅拌反应12h,收集离心并用去离子水洗涤3次,冷冻干燥得到10.2mg ZIF-8作为壳层并负载葡萄糖氧化酶和5-氟尿嘧啶的介孔普鲁士蓝材料,简称HMPB@GOx@ZIF-8@5-Fu@HA。
对比例1
采用一锅水热方法,将10mg上述HMPB与120mg聚(4-苯乙烯磺酸钠)均匀混合在40mL超纯水中,在常温的条件下以600r/min的转速搅拌反应0.5h,10000rpm/min 12min离心分离并再次超声分散在15mL超纯水中,向其中加入1mL 0.0672M的六水合硝酸锌,在常温的条件下以600r/min的转速搅拌反应0.5h,随后再向溶液中加入2mL0.1096 M的2-甲基咪唑,在常温的条件下以600r/min的转速搅拌反应0.5h,收集离心并用去离子水洗涤3次,冷冻干燥得到10.3mg ZIF-8作为壳层的介孔普鲁士蓝材料,简称HMPB@ZIF-8。
验证例
实验例1
X射线衍射实验
实验步骤:用X射线衍射仪分别对比例1所得的HMPB@ZIF-8进行X射线衍射测定。
实验结果:
由图1可知,X射线衍射曲线具有ZIF-8和HMPB两者的特征峰,表明ZIF-8在介孔普鲁士蓝表面成核生长。
实验例2
透射电子显微镜实验和元素映射实验
实验步骤:使用透射电子显微镜分别对实施例所得的HMPB、HMPB@GOx@ZIF-8@5-Fu进行透射电子显微以及对HMPB@GOx@ZIF-8@5-Fu进行元素映射实验。
实验结果:
由图2可知,HMPB表现为中空结构,在装载GOx和5-FU后,形貌和大小与HMPB无显著差异,其尺寸均在120nm左右。HMPB@GOx@ZIF-8@5-Fu包含C、N、F、Zn、P、K、Fe元素。
实验例3
x射线光电子能谱
实验步骤:取2mg粉末样品粘在双面碳导电胶或者普通双面胶,使用直接用压片机压成小片,测试样品的全谱及Fe、Zn分谱。
实验结果:
由图3(a)(b)XPS全谱及Fe分谱可知,HMPB在705和718eV处具有2p3/2和2p1/2Fe3+的峰,表明HMPB@GOx@ZIF-8@5-Fu是由铁基MOF作为核心模板。由图3(c)可知在壳层ZIF-8外延生长之后,HPGZ与HPGZF均出现了1020和1043eV处的2p3/2和2p1/2Zn2+的峰,表明核壳结构金属有机框架材料成功制备。
实验例4
产品的载药量测定
实验步骤:
取10mg实施例1的HMPB与10mg的GOx溶液混合搅拌12h,离心分离,得到上清液未负载的GOx,Bradford法利用紫外分光光度计测定595nm吸光度,随后根据GOx的标准曲线计算载药量。
取10mg实施例1的HMPB@GOx@ZIF-8与20mg的5-Fu溶液混合搅拌12h,离心分离,得到上清液未负载的5-Fu,紫外分光光度计测定256nm吸光度。随后根据5-Fu的标准曲线计算载药量。
载药量计算公式如下:
(wt%)=((m-cv))/M
其中,(wt%)为载药量,m为投入的GOx或5-Fu药物含量,mg,c为上清液浓度,mg/mL,v为上清液体积,mL,M为实施例制备的产品的总质量。
实验结果:
由图4经盐酸破坏后紫外吸收光谱图计算可知,HMPB@GOx@ZIF-8@5-Fu中GOx载药量为13.04wt%,5-FU载药量为26.02wt%。
实验例5
产品的药物及Zn2+释放量测定
实验步骤:
将实施例1的HMPB@GOx@ZIF-8@5-Fu@HA分别在pH为7.4、6.5和5.0的PBS中进行药物缓释实验。具体地,分别取2mg实施例的样品,在4mL PBS中进行缓释实验。每隔不同时间(0min,30min,60min,120min,240min,360min,1080min,1200min,1440min)吸取一定体积的上清液,离心去除可能存在的微量沉淀,随后补充所取体积对应pH的PBS,并将可能出现的沉淀移回缓释液。波长595nm测量吸光度,根据GOx的标准曲线计算药物释放量。
将实施例1的HMPB@GOx@ZIF-8@5-Fu@HA分别在pH为7.4、6.5和5.0的PBS中进行药物缓释实验。具体地,分别取2mg实施例的样品,在4mL PBS中进行缓释实验。每隔不同时间(0min,30min,60min,120min,240min,360min,1080min,1200min,1440min)吸取一定体积的上清液,离心去除可能存在的微量沉淀,随后补充所取体积对应pH的PBS,并将可能出现的沉淀移回缓释液。波长256nm测量吸光度,根据5-Fu的标准曲线计算药物释放量。
将实施例1的HMPB@GOx@ZIF-8@5-Fu@HA分别在pH为7.4、6.5和5.0的PBS或者1mg/mL的葡萄糖溶液中进行Zn2+释放实验。具体地,分别取2mg实施例的样品,在4mL PBS中进行缓释实验。每隔不同时间(0min,30min,60min,120min,240min,360min,1080min,1200min,1440min)吸取一定体积的上清液,离心去除可能存在的微量沉淀,随后补充所取体积对应pH的PBS,ICP-MS测定溶液中金属Zn的含量,根据材料中Zn的总含量计算Zn2+的释放量。
实验结果:
由图5(a)可知,GOx在被核壳材料封装具有pH相应控释性能。由图5(b)可知,5-FU封装在ZIF-8的壳层药物控释能力较差,但在pH 5.0和pH 6.5的酸性条件下药物释放较多,在中性条件药物释放较少。由图5(c)和(d)可知,Zn2+的释放受葡萄糖溶液影响极大,这得益于葡萄糖氧化酶催化葡萄糖产生酸进一步降低反应体系的pH从而加剧释放效果,表明HMPB@GOx@ZIF-8@5-Fu@HA具有葡萄糖响应的Zn2+释放特性。
实验例6
酶活性测定
实验步骤:
将实施例1中的1mg HMPB@GOx和HMPB@GOx@ZIF-8@5-Fu@HA分散在1mg/mL的葡萄糖溶液中,在不同时间点(0min、30min、60min、90min、120min、150min、180min、210min、240min)测定体系的pH值,绘制GOx消耗葡萄糖产酸后的pH变化图。
将实施例1中的HMPB@GOx@ZIF-8@5-Fu@HA分散在1mg/mL的葡萄糖溶液中配置成不同的材料浓度(0μg/mL、10μg/mL、20μg/mL、50μg/mL、100μg/mL、200μg/mL、400μg/mL),混合反应12h后,绘制GOx消耗葡萄糖含量的曲线图。
将实施例1中的HMPB@GOx@ZIF-8@5-Fu@HA分散在1mg/mL的葡萄糖溶液或PBS中配置成1mg/mL的材料浓度,混合反应6h后,使用电子顺磁共振波谱仪测定溶液中产生的·OH。
将实施例1中的HMPB@GOx@ZIF-8@5-Fu@HA分散在1mg/mL的葡萄糖溶液中配置成不同的材料浓度(0μg/mL、10μg/mL、20μg/mL、50μg/mL、100μg/mL、200μg/mL、400μg/mL),混合反应6h后,使用TMB测试溶液中·OH生成能力。
将实施例1中的HMPB@GOx@ZIF-8@5-Fu@HA分散在1mg/mL的葡萄糖溶液中配置成不同的材料浓度(0μg/mL、10μg/mL、20μg/mL、50μg/mL、100μg/mL、200μg/mL、400μg/mL)并与MB混合反应12h后,测试溶液中·OH生成能力。
实验结果:
由图6(a)可知,HMPB@GOx和HMPB@GOx@ZIF-8@5-Fu@HA均在2h左右达到酶催化反应平衡,反应体系pH能够从7.4降低到3.5左右,表明封装的GOx依旧保持酶活性。
由图6(b)可知,不同浓度的HMPB@GOx@ZIF-8@5-Fu@HA在12h内消耗葡萄糖呈现出浓度依赖,100μg/mL的材料12h足够消耗尽体系中的葡萄糖,表明封装的GOx的酶催化活性较好。
由图6(c)可知,HMPB@GOx@ZIF-8@5-Fu@HA在不同反应体系中产生·OH的情况略有不同,葡萄糖反应体系比不含葡萄糖反应体系中能够产生更多的·OH,表明反应体系中葡萄糖氧化酶消耗葡萄糖产生H2O2能进一步与Fe3+反应产生有毒的·OH。表明封装GOx后HMPB@GOx@ZIF-8@5-Fu@HA具有潜在的化学动力学性能。
进一步由图6(d)(e)可知,不同浓度的HMPB@GOx@ZIF-8@5-Fu@HA在不葡萄糖反应体系下能够产生有毒的·OH,表明封装GOx后HMPB@GOx@ZIF-8@5-Fu@HA具有浓度依赖的化学动力学性能。
实验例7
细胞毒性实验
实验步骤:
HMPB组实验溶液制备:HMPB溶于1640培养基中,并用1640培养基制备出浓度为0、0.78125、1.5625、3.125、6.25、12.5μg/mL的HMPB实验溶液;
HMPB@GOx组实验溶液制备:HMPB@GOx溶于1640培养基中,并用1640培养基制备出浓度为0、0.78125、1.5625、3.125、6.25、12.5μg/mL的HMPB@GOx实验溶液;
HMPB@GOx@ZIF-8组实验溶液制备:HMPB@GOx@ZIF-8溶于1640培养基中,并用1640培养基制备出浓度为0、0.78125、1.5625、3.125、6.25、12.5μg/mL的HMPB@GOx@ZIF-8实验溶液;
HMPB@GOx@ZIF-8@5-Fu组实验溶液制备:HMPB@GOx@ZIF-8@5-Fu溶于1640培养基中,并用1640培养基制备出浓度为0、0.78125、1.5625、3.125、6.25、12.5μg/mL的HMPB@GOx@ZIF-8@5-Fu实验溶液;
HMPB@GOx@ZIF-8@5-Fu@HA组实验溶液制备:HMPB@GOx@ZIF-8@5-Fu@HA溶于1640培养基中,并用1640培养基制备出浓度为0、0.78125、1.5625、3.125、6.25、12.5μg/mL的HMPB@GOx@ZIF-8@5-Fu@HA实验溶液;
取处于对数生长期和生长状态良好的4T1或3T3细胞,进行细胞消化,移除原培养基。先各加入2mL PBS溶液清洗两遍,加入1mL胰蛋白酶并置于培养箱中消化4min,消化完毕后,加入4mL的完全培养基(含10%胎牛血清和1%双抗)终止消化。用离心机以转速为1200r/min条件下进行离心5min,去除上清液,向其加入9mL完全培养基吹打均匀后进行细胞计算,按实验需求配制成细胞悬液并以每孔1×104个细胞种板于96孔板中,并置于培养箱中培养24h。待细胞贴壁后,将原培养基移除,每孔分别加入200μL的1640培养基溶液、HMPB实验溶液、HMPB@GOx实验溶液、HMPB@GOx@ZIF-8实验溶液、HMPB@GOx@ZIF-8@5-Fu实验溶液和HMPB@GOx@ZIF-8@5-Fu@HA实验溶液,加药完毕后,重新置于培养箱中继续培养6h。培养结束后,吸出含药培养基,每孔加入100μL的10%CCK-8试剂并置于37℃培养箱中继续培养2h。最后用酶标仪在450nm波长处测定每孔的吸光度,记录数据,利用细胞存活率计算公式求出相应细胞存活率,并以细胞存活率为y轴和药物浓度为x轴作折线图。
细胞存活率公式如下:
实验结果:
由图6(a)可知,在最大浓度12.5μg/mL下,HMPB与细胞共同孵育6小时后,只有几乎没有4T1或3T3细胞死亡,这表明HMPB具有一定的生物相容性。载药后,HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu以及HMPB@GOx@ZIF-8@5-Fu@HA对细胞呈现出明显的剂量依赖性抑制,且与3T3细胞相比对4T1细胞杀伤能力更强,这是因为4T1细胞内环境成酸性,加速葡萄糖氧化酶的催化活性,导致Zn2+和·OH的大量释放产生强细胞毒性。
实验例8
细胞摄取实验
实验步骤:
罗丹明B修饰的HMPB@GOx@ZIF-8@5-Fu@HA组实验溶液制备:取5mg的罗丹明B用10mL超纯水超声溶解,加入10mg HMPB@GOx@ZIF-8@5-Fu@HA,磁力搅拌12h。以10000r/min的速率离心10min,倒去上清液,加入超纯水洗涤,重复离心和洗涤直至上清液变无色透明,再用透析袋透析12h。最后离心,倒去上清液,冷冻干燥,即得到用罗丹明B修饰的HMPB@GOx@ZIF-8@5-Fu@HA样品。将罗丹明B修饰的HMPB@GOx@ZIF-8@5-Fu@HA溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的罗丹明B修饰的HMPB@GOx@ZIF-8@5-Fu@HA实验溶液。
取处于对数生长期和生长状态良好的4T1细胞,进行细胞消化,移除原培养基。先各加入2mL PBS溶液清洗两遍,加入1mL胰蛋白酶并置于培养箱中消化4min,消化完毕后,加入4mL的完全培养基(含10%胎牛血清和1%双抗)终止消化。用离心机以转速为1200r/min条件下进行离心5min,去除上清液,向其加入8mL完全培养基吹打均匀后进行细胞计算,按实验需求配制成细胞悬液并以每孔5×104个细胞种板于12孔板中,并置于培养箱中培养24h。待细胞贴壁后,将原培养基移除,每孔分别加入1mL的罗丹明B修饰的HMPB@GOx@ZIF-8@5-Fu@HA实验溶液,加药完毕后,重新置于培养箱中分别培养0h、1h、2h、4h、6h。培养结束后,吸出含药培养基,用1mL PBS洗涤3次,然后每孔加入1mL的4%多聚甲醛溶液室温固定细胞20min。固定完毕后,用1mL PBS洗涤2次,每孔分别加入100μL一定浓度的细胞核染料DAPI染色液1mL,37℃下避光染色15min,并用PBS洗涤2次,最后利用荧光倒置显微镜观察不同时间纳米材料的细胞吞噬情况。
实验结果:
由图8可知,随着时间的延长,4T1细胞内出现的红色荧光逐渐增亮,验证了4T1细胞对HMPB@GOx@ZIF-8@5-Fu@HA材料的有效摄取,极大程度上满足了细胞摄取材料后在细胞内释放药物的效果。
实验例9
活死细胞成像实验
实验步骤:
HMPB组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB实验溶液;
HMPB@GOx组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx实验溶液;
HMPB@GOx@ZIF-8组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8实验溶液;
HMPB@GOx@ZIF-8@5-Fu组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8@5-Fu实验溶液;
HMPB@GOx@ZIF-8@5-Fu@HA组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8@5-Fu@HA实验溶液;
取处于对数生长期和生长状态良好的4T1细胞,进行细胞消化,移除原培养基。先各加入2mL PBS溶液清洗两遍,加入1mL胰蛋白酶并置于培养箱中消化4min,消化完毕后,加入4mL的完全培养基(含10%胎牛血清和1%双抗)终止消化。用离心机以转速为1200r/min条件下进行离心5min,去除上清液,向其加入8mL完全培养基吹打均匀后进行细胞计算,按实验需求配制成细胞悬液并以每孔5×104个细胞种板于小皿中,并置于培养箱中培养24h。待细胞贴壁后,将原培养基移除,每孔分别加入200μL的1640培养基溶液、HMPB实验溶液、HMPB@GOx实验溶液、HMPB@GOx@ZIF-8实验溶液、HMPB@GOx@ZIF-8@5-Fu实验溶液和HMPB@GOx@ZIF-8@5-Fu@HA实验溶液,加药完毕后,重新置于培养箱中培养6h。培养结束后,吸出含药培养基,每孔加入100μL一定浓度的Calcein-AM(染活细胞为绿色)和PI(染死细胞为红色)的混合荧光染料,37℃下避光染色15min,并用PBS洗涤2次,置于荧光显微镜下观察细胞荧光成像情况。
实验结果:
由图9可知,与HMPB、HMPB@GOx两组相比,HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu、HMPB@GOx@ZIF-8@5-Fu@HA表现出有效的细胞杀伤效果,从染色的死亡细胞发出强烈的红色荧光可以看出,这是由于GOx、Zn2+和5-FU的协同抗癌作用所致。
实验例10
细胞内ROS成像实验
实验步骤:
HMPB组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB实验溶液;
HMPB@GOx组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx实验溶液;
HMPB@GOx@ZIF-8组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8实验溶液;
HMPB@GOx@ZIF-8@5-Fu组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8@5-Fu实验溶液;
HMPB@GOx@ZIF-8@5-Fu@HA组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8@5-Fu@HA实验溶液;
取处于对数生长期和生长状态良好的4T1细胞,进行细胞消化,移除原培养基。先各加入2mL PBS溶液清洗两遍,加入1mL胰蛋白酶并置于培养箱中消化4min,消化完毕后,加入4mL的完全培养基(含10%胎牛血清和1%双抗)终止消化。用离心机以转速为1200r/min条件下进行离心5min,去除上清液,向其加入8mL完全培养基吹打均匀后进行细胞计算,按实验需求配制成细胞悬液并以每孔5×104个细胞种板于小皿中,并置于培养箱中培养24h。待细胞贴壁后,将原培养基移除,每孔分别加入200μL的1640培养基溶液、HMPB实验溶液、HMPB@GOx实验溶液、HMPB@GOx@ZIF-8实验溶液、HMPB@GOx@ZIF-8@5-Fu实验溶液和HMPB@GOx@ZIF-8@5-Fu@HA实验溶液,加药完毕后,重新置于培养箱中培养6h。培养结束后,吸出含药培养基,按照试剂盒测试要求配制1:1000的DCFH-DA探针,每孔添加探针200μL,37℃孵育30min,PBS洗涤两遍,倒置荧光显微镜记录ROS生成量。
实验结果:
由图10可知,与HMPB、HMPB@GOx两组相比,HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu、HMPB@GOx@ZIF-8@5-Fu@HA表现出强烈的细胞内ROS聚集效果,这证明了ZIF-8在细胞内解离释放Zn2+可以引起强烈的ROS杀伤细胞,起到化学动力学治疗效果。
实验例11
线粒体膜电位去极化实验
实验步骤:
HMPB组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB实验溶液;
HMPB@GOx组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx实验溶液;
HMPB@GOx@ZIF-8组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8实验溶液;
HMPB@GOx@ZIF-8@5-Fu组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8@5-Fu实验溶液;
HMPB@GOx@ZIF-8@5-Fu@HA组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8@5-Fu@HA实验溶液;
取处于对数生长期和生长状态良好的4T1细胞,进行细胞消化,移除原培养基。先各加入2mL PBS溶液清洗两遍,加入1mL胰蛋白酶并置于培养箱中消化4min,消化完毕后,加入4mL的完全培养基(含10%胎牛血清和1%双抗)终止消化。用离心机以转速为1200r/min条件下进行离心5min,去除上清液,向其加入8mL完全培养基吹打均匀后进行细胞计算,按实验需求配制成细胞悬液并以每孔5×104个细胞种板于小皿中,并置于培养箱中培养24h。待细胞贴壁后,将原培养基移除,每孔分别加入200μL的1640培养基溶液、HMPB实验溶液、HMPB@GOx实验溶液、HMPB@GOx@ZIF-8实验溶液、HMPB@GOx@ZIF-8@5-Fu实验溶液和HMPB@GOx@ZIF-8@5-Fu@HA实验溶液,加药完毕后,重新置于培养箱中培养6h。培养结束后,吸出含药培养基,按照JC-1线粒体膜电位检测试剂盒说明配制JC-1工作液每孔添加1mL JC-1试剂,37℃避光孵育20min,使用JC-1洗涤液将细胞洗涤两次去除染料,再加入0.5mL全培,同时采用DAPI染料对细胞核进行染色,倒置荧光显微镜记录荧光变化。
实验结果:
由图11可知,与HMPB、HMPB@GOx两组相比,HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu、HMPB@GOx@ZIF-8@5-Fu@HA表现出强烈的线粒体膜损伤,这也证明了ZIF-8在细胞内解离释放Zn2+可以引起线粒体损伤致使癌细胞死亡。
实验例12
流式细胞凋亡实验
实验步骤:
HMPB组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB实验溶液;
HMPB@GOx组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx实验溶液;
HMPB@GOx@ZIF-8组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8实验溶液;
HMPB@GOx@ZIF-8@5-Fu组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8@5-Fu实验溶液;
HMPB@GOx@ZIF-8@5-Fu@HA组实验溶液制备HMPB溶于1640培养基中,并用1640培养基制备出浓度为12.5μg/mL的HMPB@GOx@ZIF-8@5-Fu@HA实验溶液;
取处于对数生长期和生长状态良好的4T1细胞,进行细胞消化,移除原培养基。先各加入2mL PBS溶液清洗两遍,加入1mL胰蛋白酶并置于培养箱中消化4min,消化完毕后,加入4mL的完全培养基(含10%胎牛血清和1%双抗)终止消化。用离心机以转速为1200r/min条件下进行离心5min,去除上清液,向其加入8mL完全培养基吹打均匀后进行细胞计算,按实验需求配制成细胞悬液并以每孔5×104个细胞种板于小皿中,并置于培养箱中培养24h。待细胞贴壁后,将原培养基移除,每孔分别加入200μL的1640培养基溶液、HMPB实验溶液、HMPB@GOx实验溶液、HMPB@GOx@ZIF-8实验溶液、HMPB@GOx@ZIF-8@5-Fu实验溶液和HMPB@GOx@ZIF-8@5-Fu@HA实验溶液,加药完毕后,重新置于培养箱中培养6h。培养结束后,吸出含药培养基,收集细胞,5μL Annexin V-FITC和10μLPI常温避光染色15min,采用流式细胞仪(CytoFLEX)进行凋亡定量。
实验结果:
由图12可知,与HMPB、HMPB@GOx两组相比,HMPB@GOx@ZIF-8、HMPB@GOx@ZIF-8@5-Fu、HMPB@GOx@ZIF-8@5-Fu@HA表现出强烈的细胞凋亡效应,这证明了HMPB@GOx@ZIF-8@5-Fu@HA是具有非常有效的抗癌效果。
从上述实验结果可以看出,本发明利用孔道分级制备的负载两种抗癌药物的核壳结构金属有机框架材料具有较高的GOx负载能力(13.04wt%)和5-FU负载能力(26.02wt%)。同时通过合理选择ZIF-8来划分HMPB的介孔孔道,使其具备葡萄糖、pH响应共释能力,可以实现GOx、Zn2+和5-氟尿嘧啶协同抗癌作用,增强了对癌症组织部位的治疗效果,具有一定的临床应用潜能。
Claims (8)
1.一种兼具大孔和微孔尺寸的核壳结构金属有机框架复合材料,其特征在于,所述框架复合材料的分子式为HMPB@GOx@ZIF-8@5-FU@HA;所述负载的抗癌药物为葡萄糖氧化酶和5-氟尿嘧啶;其中复合材料中葡萄糖氧化酶被封装在HMPB介孔内,ZIF-8薄层生长在HMPB表面,所述5-氟尿嘧啶吸附进入ZIF-8壳层内。
2.根据权利要求1所述的核壳结构金属有机框架复合材料,其特征在于,所述葡萄糖氧化酶载药量可以达到13.04wt%,5-氟尿嘧啶载药量可以达到26.02wt%。
3.根据权利要求1所述的核壳结构金属有机框架复合材料,其特征在于,所述HMPB为核心结构,是以聚乙烯吡咯烷酮为配体,铁离子为金属离子利用水热法合成的纳米尺寸的金属有机骨架材料;所述ZIF-8为壳层结构,是以2-甲基咪唑为配体,锌离子为金属离子,以外延生长法在HMPB表面原位生长。
4.一种权利要求1-3任一项所述的核壳结构金属有机框架复合材料的制备方法,其特征在于,采用以下步骤:
(1)合成立方体普鲁士蓝纳米颗粒,盐酸刻蚀形成中空介孔结构的HMPB;
(2)将步骤(1)制备的中空介孔结构的HMPB与葡萄糖氧化酶溶液一同搅拌得到金属有机框架复合材料HMPB@GOx;
(3)将步骤(2)制备的HMPB@GOx分散于聚(4-苯乙烯磺酸钠)溶液进行改性,将改性后的HMPB@GOx分散于水溶液中,依次向其中加入硝酸锌溶液和2-甲基咪唑溶液,搅拌后获得核壳结构金属有机框架复合材料HMPB@GOx@ZIF-8;
(4)将步骤(3)制备的HMPB@GOx@ZIF-8分散于5-氟尿嘧啶和透明质酸的水溶液中搅拌,得到负载葡萄糖氧化酶和5-氟尿嘧啶的核壳结构金属有机框架复合材料HMPB@GOx@ZIF-8@5-FU@HA。
5.根据权利要求3所述的制备方法,其特征在于,所述步骤(1)所述的立方体普鲁士蓝纳米颗粒采用水热法制得;具体步骤为:
(1)将铁氰化钾和聚乙烯吡咯烷酮超声溶解在0.1mM的盐酸溶液中,80℃水热反应24h后收集产物、洗涤,得到立方体普鲁士蓝纳米颗粒;
(2)将步骤(1)制备的立方体普鲁士蓝纳米颗粒与聚乙烯吡咯烷酮超声分散于1mM的盐酸溶液中,140℃下反应2h,反应结束后收集产物、洗涤,得立方体普鲁士蓝纳米颗粒。
6.根据权利要求3所述的制备方法,其特征在于,所述步骤(2)所述的金属有机框架复合材料HMPB@GOx的制备方法:
将10mg立方体普鲁士蓝纳米颗粒与10mg葡萄糖氧化酶在10mL超纯水中超声10min并500r/min搅拌12h,反应结束后收集产物、洗涤,得HMPB@GOx。
7.根据权利要求3所述的制备方法,其特征在于,所述步骤(3)所述核壳结构金属有机框架复合材料HMPB@GOx@ZIF-8的制备方法为:
将10mg HMPB@GOx与120mg聚(4-苯乙烯磺酸钠)在30mL超纯水中超声10min并500r/min搅拌30min,反应结束后离心获得沉淀并再次分散在15mL超纯水中,向其中投入1mL 0.067M的六水合硝酸锌搅拌500r/min搅拌30min,再次向其中加入2mL0.1096M的二甲基咪唑500r/min搅拌30min,反应结束后收集产物、洗涤,得HMPB@GOx@ZIF-8。
8.根据权利要求3所述的制备方法,其特征在于,所述步骤(4)所述的核壳结构金属有机框架复合材料HMPB@GOx@ZIF-8@5-Fu@HA;具体步骤为:将10mgHMPB@GOx@ZIF-8与20mg的5-氟尿嘧啶以及15mg的HA分散于10mL的超纯水中,常温下500r/min搅拌12h,反应结束后收集产物、洗涤,得HMPB@GOx@ZIF-8@5-Fu@HA。
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