CN116350800A - 葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒的制备与应用 - Google Patents
葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒的制备与应用 Download PDFInfo
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Abstract
本发明公开了一种葡萄糖氧化酶‑金属‑姜黄素自组装纳米颗粒及其制备与应用,属于纳米材料制备和生物医学应用领域。其是将GOx、Cu2+、姜黄素作为自组装单元,利用配位协同疏水作用力驱动三者在纳米尺度进行自组装,获得自组装颗粒,从而实现GOx的高负载(54%),同时,基于Cu2+对GOx酶活性的抑制竞争作用,可实现pH精准调控酶活性,使得饥饿疗法选择性“开启”和“关闭”,再通过在自组装颗粒表面修饰靶分子RGD,进一步增强自组装颗粒对正常细胞和癌细胞的区分化抑制效果,使所得自组装纳米颗粒在很低的GOx剂量下(1.43μg/ml),仅依靠单一的饥饿疗法就可实现对肿瘤细胞的高抑制(75%)。
Description
技术领域
本发明属于纳米材料制备和生物医学应用领域,具体涉及一种葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒及其制备方法与应用。
背景技术
饥饿疗法(ST)是指通过阻断血流或消耗其必需营养物质和能量供应来抑制肿瘤生长和增殖的有效方法。其中,基于葡萄糖氧化酶(GOx)介导设计的阻断肿瘤细胞内葡萄糖供应策略在肿瘤抑制方面已引起广泛关注。葡萄糖是肿瘤生长和增殖的主要能量供应者,GOx可以特异性催化葡萄糖转化为葡萄糖酸和H2O2,通过直接消耗葡萄糖致使癌细胞缺乏能量的提供,从而诱导癌细胞死亡。同时,生成的H2O2会促进芬顿反应的进程,大幅提高细胞内羟基自由基(•OH)的生成量,对癌细胞造成进一步严重的氧化损伤。因此,近年来通过将GOx介导的肿瘤饥饿治疗与其他肿瘤治疗手段整合,比如饥饿疗法协同低氧激活疗法、氧化疗法、光动力疗法和光照疗法等多模式协同抗癌疗法变成重要的研究热点。
但是,目前基于GOx的肿瘤疗法仍处于起步阶段,距离其临床应用还存在许多急需解决的问题:一是由于葡萄糖和氧的底物在体内无处不在,因此非特异性GOx催化不可避免地会引起其它不良副作用,例如糖果症、组织缺氧和反应性氧(ROS)生成,造成对正常组织的极大损害;二是GOx在体内运输过程中,很容易被蛋白酶降解,从而失活,同时还存在药物吸收不良、生物利用度差、体内代谢快等缺点;三是目前GOx主要是通过设计各种载体负载运输到体内,普遍存在负载率低(<25%)、药物使用剂量大等缺点。以上问题都严重阻碍了GOx介导的饥饿疗法的临床应用。
发明内容
针对现有技术存在的问题,本发明提供了一种葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒及其制备方法与应用,该自组装纳米颗粒具有高GOx负载率(54%)、pH精准调控GOx酶活性及主动靶向肿瘤组织等性能特征,能实现主要依赖于GOx的饥饿疗法的高效肿瘤抑制效果。
为实现上述目的,本发明采用如下技术方案:
一种葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒的制备方法,其是使葡萄糖氧化酶(GOx)、铜离子(Cu2+)与姜黄素(Cur)经自组装后,在其表面修饰Arg-Gly-Asp肽(RGD)而制成;其具体包括以下步骤:
1)将GOx充分溶解到超纯水中,得到GOx溶液;
2)将Cu2+溶解到去离子水中,得到Cu2+溶液;
3)将Cur用有机溶剂充分溶解,得到Cur溶液;
4)在搅拌条件下,在步骤1)所得GOx溶液中加入适量Tris-HCl缓冲液调节pH,室温搅拌10min后,同时加入步骤2)所得的Cu2+溶液和步骤3)所得的Cur溶液,反应5~30分钟后,经10000 rpm高速离心20min,取上清液置于透析袋(WMCO 300kDa)中,用磷酸缓冲溶液透析24小时(每8小时换一次透析液),随后将透析后上清液超滤纯化,得到自组装颗粒GOx-Cu2+-Cur NPs(GCC NPs);
5)将步骤4)所得GCC NPs加入EDC及NHS活化后,再加入RGD反应过夜,反应液经超滤后获得所述自组装纳米颗粒RGD-GOx-Cu2+-Cur NPs。
进一步地,步骤3)中所述有机溶剂包括二甲亚砜、无水乙醇、无水丙酮中的任意一种。
进一步地,步骤4)中搅拌的转速为900 rpm。
进一步地,步骤4)中调节pH的范围为8~9。
进一步地,步骤4)中GOx溶液、Cu2+溶液与Cur溶液的用量按GOx、Cu2+和Cur的质量比为1.5:(0.15~2):(1~4)进行换算。
进一步地,步骤4)所得自组装颗粒为球形单分散颗粒,其尺寸为40~45 nm。
进一步地,步骤5)中RGD的用量与所用GOx的质量比为0.2:1.5。
进一步地,步骤5)所得葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒的粒径为50~55 nm,其具有高GOx负载率、pH精准调控GOx酶活性及主动靶向肿瘤组织等性能特征。
所述的葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒可通过pH精准调控GOx酶活性,使得饥饿疗法选择性“开启”和“关闭”,实现可调控饥饿疗法抑制肿瘤细胞的生长,因而可用于制备抗肿瘤药物。
本发明通过将葡萄糖氧化酶(GOx)、铜离子(Cu2+)、姜黄素(Cur)作为自组装单元,利用配位协同疏水作用力驱动三者在纳米尺度进行自组装,获得自组装颗粒,从而实现GOx的高负载(54%)。同时,Cu2+对GOx具有强烈的酶活性抑制作用,在正常生理条件下Cu2+可占据酶的活性位点,有效抑制GOx的活性;而在肿瘤部位弱酸环境下,Cu2+被剥离,GOx的活性恢复,因而可以通过少量Cu2+对GOx的抑制竞争作用,实现pH精准调控酶活性,使得饥饿疗法选择性“开启”和“关闭”。而靶分子RGD的表面修饰,可以进一步增强自组装颗粒对正常细胞和癌细胞的区分化抑制效果,因而所得自组装纳米颗粒在很低的GOx剂量下(1.43μg/ml),仅依靠单一的饥饿疗法就可实现对肿瘤细胞的高抑制(75%)。
本发明的显著优点在于:
(1)本发明通过将GOx作为自组装基本单元(而不是吸附负载),有效实现了GOx的高负载(54.43%)。
(2)本发明通过Cu2+与GOx的配位络合,利用Cu2+对GOx酶活性的抑制竞争作用,可实现pH精准调控酶活性,使得饥饿疗法选择性“开启”和“关闭”。
(3)本发明所得自组装纳米颗粒可在低GOx剂量(1.43μg/ml)下,实现通过饥饿疗法对肿瘤的高效抑制效率,可有效避免高药物剂量使用带来的副作用。
附图说明
图1为实施例所制得GOx-Cu2+-Cur NPs与RGD-GOx-Cu2+-Cur NPs的粒径图(A)及电位图(B)。
图2为实施例所制备GOx-Cu2+-Cur NPs(A)与RGD-GOx-Cu2+-Cur NPs(B)的TEM图及粒径分布图。
图3为实施例所制备RGD-GOx-Cu2+-Cur NPs在PBS(A)与DMEM(B)中14天内的粒径变化图。
图4为实施例所制备RGD-GOx-Cu2+-Cur NPs与GOx在不同pH条件下与10mM葡萄糖溶液孵育不同时间(6、12、18、24h)产生H2O2的对比情况图。
图5为实施例所制备RGD-GOx-Cu2+-Cur NPs在不同pH条件下与不同浓度葡萄糖溶液(1、2、4、6、8、10mM)孵育24h后H2O2产量的对比情况图。
图6为实施例所制备RGD-GOx-Cu2+-Cur NPs受pH刺激响应Cu2+(A)和Cur(B)的释放曲线图。
图7为实施例所制备RGD-GOx-Cu2+-Cur NPs对LO2正常细胞的生物相亲性表征图。
图8为实施例所制备RGD-GOx-Cu2+-Cur NPs对HepG2癌细胞生长的抑制效果图。
图9为LO2细胞(6h)及HepG2细胞(2、4、6h)对RGD-GOx-Cu2+-Cur NPs摄取情况的共聚焦图。
图10为实施例所制备RGD-GOx-Cu2+-Cur NPs在HepG2细胞内产生ROS的共聚焦荧光图。
图11为实施例所制备RGD-GOx-Cu2+-Cur NPs处理HepG2后,经AM-PI染色的癌细胞共聚焦荧光图。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
实施例
1)称取10.0 mg的GOx溶于1.0 mL超纯水,配制为10.0 mg/mL的GOx溶液;
2)称取1.705mg的CuCl2·2H2O溶于1.0mL超纯水中,配制为0.01M的Cu2+溶液;
3)称取5mg的Cur溶于1.0 mL二甲基亚砜(DMSO)中,配制为5.0 mg/mL的Cur溶液;
4)称取25.0mg的RGD溶于1.0mL超纯水中,配制成25.0 mg/mL的RGD溶液;
5)在反应瓶中加入150 μL GOx溶液,然后加入55 μL 0.1M Tris-HCl缓冲液调节pH为8,在室温条件下900 rpm搅拌10min,随后加入100 μL Cu2+溶液和200μL Cur溶液,继续搅拌15 min后,取出反应液在10000 rpm条件下离心20min,取上清液置于透析袋(WMCO300kDa)中,用磷酸缓冲溶液透析24小时(每8小时换一次透析液),随后将透析后上清液超滤纯化,即得到GOx-Cu2+-Cur NPs(GCC NPs);
6)在2 mg/mL的GCC NPs中加入20 μL 5 mg/mL EDC溶液和150 μL 20 mg/mL NHS溶液,最后加入8μL RGD溶液,反应过夜,超滤水洗两遍,即得到RGD-GOx-Cu2+-Cur NPs。
应用实施例
1. 将实施例所得RGD-GOx-Cu2+-Cur NPs与GOx-Cu2+-Cur NPs分别加入到纯水中,测其粒径和电位,结果如图1所示。
由图1可见,RGD-GOx-Cu2+-Cur NPs与GOx-Cu2+-Cur NPs的粒径为分别为58.77 nm与43.76nm(a),电位分别为-8.75 mV与-19.4mV(b)。
2. 将实施例制备好的GOx-Cu2+-Cur NPs与RGD-GOx-Cu2+-Cur NPs充分稀释,用移液枪分别吸取10μl滴加于干净的超薄碳膜上,待样品干燥后,将超薄碳膜送入样品室,在加速电压为200 kv的条件下,对纳米颗粒进行形貌表征,并使用软件统计两种纳米颗粒的粒径,结果如图2所示。
由图2可见,GOx-Cu2+-Cur NPs的粒径约为39~46 nm,RGD-GOx-Cu2+-Cur NPs的粒径约为46~56 nm,两种纳米颗粒的尺寸均一且分散性良好。
3. 将实施例得到的RGD-GOx-Cu2+-Cur NPs分别加入到PBS和DMEM培养液中封口保存,取样测量其粒径变化,结果如图3所示。
由图3可见,两周内纳米颗粒在PBS和DMEM培养液中的粒径没有发生很大的变化,表明RGD-GOx-Cu2+-Cur NPs具有良好的分散性和稳定性。
4. 为了评估RGD-GOx-Cu2+-Cur NPs的pH响应催化活性,将制备好的RGD-GOx-Cu2 +-CurRGCC NPs或者葡萄糖氧化酶分别与葡萄糖水溶液混合,并分别浸入pH7.4或5.5的PBS缓冲溶液中,使得葡萄糖的浓度均为10 mM,反应6 h、12 h、18 h、24 h后,摇匀,用移液枪吸取100μL反应液,用过氧化氢检测试剂盒检测反应后的过氧化氢含量,结果如图4所示。
由图4可见,RGD-GOx-Cu2+-Cur NPs在pH 5.5的PBS缓冲液中孵育,随时间增加产生过氧化氢量大大增加,而在pH 7.4的PBS缓冲液产生过氧化氢量则是增加缓慢,这证实了在pH 7.4的生理环境下,RGD-GOx-Cu2+-Cur NPs仅具有低催化活性,而在微酸环境下其催化活性会被重新激活。
为研究不同浓度葡萄糖对RGD-GOx-Cu2+-Cur NPs催化活性释放的影响,将等量RGD-GOx-Cu2+-Cur NPs与不同浓度(1、2、4、6、8、10mM)的葡萄糖水溶液混合,并浸入pH 7.4或5.5的PBS(10 mM)缓冲溶液中,反应24 h后,摇匀,用移液枪吸取100μL反应液,根据过氧化氢含量检测试剂盒标准步骤检测过氧化氢含量,结果如图5所示。
由图5可见,RGD-GOx-Cu2+-Cur NPs在pH 5.5的PBS缓冲液中,过氧化氢产生量随着葡萄糖浓度的增加而显著增加。而RGD-GOx-Cu2+-Cur NPs在pH 7.4的PBS缓冲液中,过氧化氢仅有微弱产生,这主要是因为在碱性条件下,铜离子占据了葡萄糖氧化酶的活性位点,使得即使有大量的葡萄糖存在,葡萄糖氧化酶也无法有效催化葡萄糖。
5. 为进一步研究RGD-GOx-Cu2+-Cur NPs受pH响应释放催化活性的原理,将制备的RGD-GOx-Cu2+-Cur NPs转移到透析袋(MWCO 8000 Da),并浸入pH 7.4或5.5的PBS(10 mM)中,在室温黑暗中缓慢搅拌48 h,并在透析0 h、1 h、3 h、5 h、7 h、9 h、12 h、16 h、20 h、24h、30 h、36 h、48 h时,取出缓冲液并用新鲜PBS缓冲液补足,用ICP-AES法测定Cu2+的含量。同时,将RGD-GOx-Cu2+-Cur NPs转移到透析袋(MWCO 8000 Da),并浸入含5%吐温80的PBS(10mM,pH7.4或5.5)缓冲溶液中,在室温黑暗中缓慢搅拌48 h,并在透析0 h、1 h、3 h、5 h、7h、9 h、12 h、16 h、20 h、24 h、30 h、36 h、48 h时,取出缓冲液并用新鲜PBS缓冲液补足,用紫外-可见分光光度法测定Cur的含量,结果由图6所示。
由图6可见,pH 5.5时Cu2+与Cur释放量远大于pH 7.4时的释放量,这表明Cu2+与Cur在酸性条件下可以进行响应性释放。
6. 为了考察RGD-GOx-Cu2+-Cur NPs的生物相亲性,将RGD-GOx-Cu2+-Cur NPs和人正常肝细胞(LO2细胞)共孵育,判断RGD-GOx-Cu2+-Cur NPs的毒性情况。待LO2细胞生长至90%左右进行细胞传代操作,其操作步骤如下:去掉培养瓶中的旧培养基,然后加入2.0 mLPBS溶液清洗3次,之后加入1.0 mL胰蛋白酶消化细胞1分钟左右,去除胰酶,加入2.0 mLDMEM培养基停止消化并轻柔吹打细胞使细胞悬浮得到细胞原液,取1/4的细胞原液分装入新的培养瓶中,置于37℃,5% CO2培养箱中继续培养。取出一部分LO2细胞原液用DMEM培养液稀释使其密度为105个/mL,并以每孔104个细胞接种至96孔板中,置于37℃,5% CO2的培养箱中培养24小时后,移去孔板中旧培养液,加入GOx浓度依次为0、0.55、0.77、0.99、1.21、1.43μg/mL的RGD-GOx-Cu2+-Cur NPs培养液,每个浓度设置4个重复孔。培养6小时后,吸出培养液并用PBS清洗2次,加入100 μL新鲜培养液继续培养18小时后,每孔加入10 μL浓度为5.0 mg/mL的MTT,孵育4~6小时,小心移去培养液,加入150 μL DMSO,放入恒温摇床中在37℃、150 rpm条件下培养15分钟,然后用酶标仪测量各孔溶液在490nm处的吸收值,计算存活率,以评价RGD-GOx-Cu2+-Cur NPs的生物相亲性,结果如图7所示。
由图7可见,RGD-GOx-Cu2+-Cur NPs在GOx浓度为1.43 μg/mL时,细胞存活率在80%以上,说明RGD-GOx-Cu2+-Cur NPs的生物相亲性好。
同时,为了考察RGD-GOx-Cu2+-Cur NPs的体外治疗效果,将RGD-GOx-Cu2+-Cur NPs和肝癌细胞(HepG2细胞)共孵育,判断RGD-GOx-Cu2+-Cur NPs的毒性情况。其操作步骤为:首先,将传代过程中的HepG2细胞悬液用培养基重新分散,并使得细胞密度为5×104个/mL,将重新分散的细胞悬液接种于96孔板中,每组设置四个平行孔,每孔100 μL,然后置于37 ℃,5% CO2的细胞培养箱培养24 h,使得细胞完全贴壁。去除旧的培养液,分别向各孔加入GOx浓度依次为0 μg/mL、0.55μg/mL、0.77 μg/mL、0.99μg/mL、1.21 μg/mL、1.43μg/mL的RGD-GOx-Cu2+-Cur NPs溶液(DMEM培养基做溶剂,葡萄糖浓度为10 mM)继续培养4 h,吸除RGD-GOx-Cu2+-Cur NPs溶液,用PBS洗涤细胞三遍,加入新的培养液培养20 h,每孔加入10 µL 5mg/mL的MTT溶液,移入培养箱继续培养4 h,小心吸除培养液,防止蓝紫色甲瓒结晶被吸出,每孔加入150 µL DMSO,最后在37 ℃恒温摇床避光震荡15 min,等待蓝紫色甲瓒结晶全部溶解后,用酶标仪测定每孔在490 nm处的吸光度,计算细胞的存活率,结果如图8所示。
由图8可见,RGD-GOx-Cu2+-Cur NPs仅在GOx浓度为1.43 μg/mL时,细胞死亡率就达到70%以上,说明RGD-GOx-Cu2+-Cur NPs的体外实验治疗效果十分优异。
7. 通过激光扫描共聚焦显微镜比较LO2细胞和HepG2细胞对RGD-GOx-Cu2+-CurNPs的摄取来探究RGD-GOx-Cu2+-Cur NPs对癌细胞的靶向能力。其操作步骤为:将细胞密度为1×104个/mL的LO2细胞和HepG2细胞分别接种于共聚焦培养皿中,每孔400 µL,置于37℃,5% CO2的细胞培养箱培养24 h,使得细胞完全贴壁。弃去培养液,加入含罗丹明B标记的RGD-GOx-Cu2+-Cur NPs的无血清培养基,置于细胞培养箱培养LO2细胞6 h,HepG2细胞分别培养2、4、6 h,弃去培养基,用PBS洗涤细胞两次,加入培养基,将共聚焦培养皿置于激光共聚焦显微镜上拍摄激发波长为488 nm的荧光图谱,结果如图9所示。
由图9可见,在2h时,HepG2细胞对RGD-GOx-Cu2+-Cur NPs的摄取情况与LO2细胞6h时相似。在6h时,HepG2对RGD-GOx-Cu2+-Cur NPs的细胞摄取量远大于LO2细胞,说明对RGD-GOx-Cu2+-Cur NPs对HepG2癌细胞的靶向能力更强。
8. 通过DCFH-DA探针来检测RGD-GOx-Cu2+-Cur NPs在HepG2细胞内产生ROS的含量。其操作步骤为:将细胞密度为1×104个/mL的HepG2细胞接种于共聚焦培养皿中,置于37℃,5% CO2的细胞培养箱培养24 h,使得细胞完全贴壁。弃去培养液,将阴性对照组设置为无血清培养基培养的细胞,阳性对照组设置为含0.1 mM H2O2的无血清培养基培养的细胞,实验组分别加入含GOx浓度为2.2µg/mL的RGD-GOx-Cu2+-Cur NPs和GOx-Cu2+-Cur NPs的无血清培养基,继续培养6 h后,均移除旧的培养液,用PBS洗涤细胞一遍,然后在各组细胞中加入DCFH-DA探针孵育30 min,最后用PBS轻洗涤细胞两遍,加入无血清培养基,将共聚焦培养皿放置于激光共聚焦显微镜上拍摄激发波长为488 nm的荧光图谱,结果如图10所示。
由图10可见,RGD-GOx-Cu2+-Cur NPs产生的荧光与阳性对照组相近。而GOx-Cu2+-Cur NPs由于缺少靶向功能,其ROS产生的绿色荧光明显低于RGD-GOx-Cu2+-Cur NPs。
9. 为了探究RGD-GOx-Cu2+-Cur NPs对HepG2细胞的具体杀伤情况,通过激光扫描共聚焦显微镜拍摄Calcein-AM/PI荧光探针染色的活/死细胞的共聚焦图。其操作步骤为:将细胞密度为5×104个/mL的HepG2细胞接种于共聚焦培养皿中,置于37 ℃,5% CO2的细胞培养箱培养24 h,使得细胞完全贴壁。弃去培养液,将阴性对照组设置为无血清培养基培养的细胞,实验组分别加入含GOx浓度为2.2µg/mL的RGD-GOx-Cu2+-Cur NPs和GOx-Cu2+-CurNPs的无血清培养基,继续培养4 h后,均移除旧的培养液,用PBS洗涤细胞两遍,然后各组加入无血清新鲜培养液孵育20 h,均移除旧的培养液,加入Calcein-AM/PI 荧光染料孵育40min,用PBS轻洗涤细胞两遍,加入无血清培养基,通过激光扫描共聚焦显微镜成像,结果如图11所示。
由图11可见,对照组几乎不存在死细胞所产生的红色荧光;GOx-Cu2+-Cur NPs组红色荧光与绿色荧光并存,说明GOx-Cu2+-Cur NPs对HepG2细胞有一定的杀伤作用;而RGD-GOx-Cu2+-Cur NPs组存在大量红色荧光和微量绿色荧光,说明RGD-GOx-Cu2+-Cur NPs的体内实验治疗效果显著。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (9)
1.一种葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒的制备方法,其特征在于,所述自组装纳米颗粒是使葡萄糖氧化酶、铜离子与姜黄素经自组装后,在其表面修饰RGD肽而制成。
2.根据权利要求1所述的葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒的制备方法,其特征在于,具体包括以下步骤:
1)将葡萄糖氧化酶、铜离子与姜黄素分别溶解,得到相应溶液;
2)将步骤1)所得溶液按比例混合,调pH后反应一段时间,再经离心,取上清液透析,获得自组装颗粒;
3)将步骤2)所得自组装颗粒加入EDC及NHS活化后,再加入RGD肽反应过夜,经超滤后获得所述自组装纳米颗粒。
3.根据权利要求2所述的葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒的制备方法,其特征在于,步骤2)中按葡萄糖氧化酶、铜离子与姜黄素的质量比为1.5:(0.15~2):(1~4)将相应溶液进行混合。
4.根据权利要求2所述的葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒的制备方法,其特征在于,步骤2)中调节pH的范围为8~9。
5.根据权利要求2所述的葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒的制备方法,其特征在于,步骤2)中所述反应的时间为5~30分钟。
6.根据权利要求2所述的葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒的制备方法,其特征在于,步骤2)所得自组装颗粒为球形单分散颗粒,其尺寸为40~45 nm。
7.根据权利要求2所述的葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒的制备方法,其特征在于,步骤3)中RGD肽的用量与所用葡萄糖氧化酶的质量比为0.2:1.5。
8.一种如权利要求1-7任一方法制备的葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒,其特征在于,所述葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒具有高GOx负载率、pH精准调控GOx酶活性及主动靶向肿瘤组织的性能特征,其粒径为50~55 nm。
9.一种如权利要求8所述的葡萄糖氧化酶-金属-姜黄素自组装纳米颗粒在制备抗肿瘤药物中的应用。
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