CN114732795B - 一种长循环多功能金属有机框架纳米制剂的制备方法 - Google Patents
一种长循环多功能金属有机框架纳米制剂的制备方法 Download PDFInfo
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- CN114732795B CN114732795B CN202210003438.XA CN202210003438A CN114732795B CN 114732795 B CN114732795 B CN 114732795B CN 202210003438 A CN202210003438 A CN 202210003438A CN 114732795 B CN114732795 B CN 114732795B
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Abstract
本发明涉及一种长循环多功能金属有机框架纳米制剂的制备方法,该方法先以PVP为矿化剂制得粒径适中的GA/Fe纳米复合物,然后获得白色纳米颗粒;将白色纳米颗粒溶解,浸泡于透明质酸溶液中进行孵育,制得多功能金属有机框架纳米制剂;该制剂具有优异的生物相容性、肿瘤靶向能力、双模态成像功能和联合CDT/饥饿治疗/化疗的能力,可以同时实现肿瘤原位成像和治疗,由于聚乙二醇的掺杂可以在体内实现隐身,实现长循环,具有良好的靶向性,封装基于聚乙烯吡咯烷酮GA/Fe纳米复合物使该制剂同时实现基于Fe3+的T2成像效果,可以准确获得肿瘤空间位置,没食子酸可还原芬顿反应产生的Fe3+,加速芬顿反应产生·OH有效杀死肿瘤细胞;在肿瘤治疗中具有良好的应用前景。
Description
技术领域
本发明涉及一种长循环多功能金属有机框架纳米制剂的制备方法,属于生物医药技术领域。
背景技术
随着环境的恶化和生活方式的改变,癌症的发病率逐年升高,预计在未来20年内新诊断的癌症病例数量将上升约70%。因此恶性肿瘤仍然是我们全人类面临的难题,安全有效的治疗手段仍然亟待被开发和应用。
近年来,化学动力学疗法(CDT)作为一种新兴的癌症治疗策略受到广泛关注。内源性过氧化氢(H2O2)通过金属离子(主要是Fe(III)和Fe(II))介导的芬顿反应可以转化为具有细胞毒性的羟基自由基(·OH),促使细胞内脂质过氧化物堆积,最终诱导细胞发生铁死亡。然而,生理条件H2O2的含量有限,在一定程度上限制了CDT的应用。利用葡萄糖氧化酶(GOx)催化葡萄糖(GO)生成葡萄糖酸和H2O2,可以满足CDT对H2O2的需要。另外,葡萄糖氧化酶GOx在反应过程中可以消耗细胞内GO和O2,引起肿瘤细胞内维持生长和代谢的能量不足,最终导致细胞肿瘤死亡。该方法被称为饥饿疗法。目前研究者通常利用Fe(II)来引发芬顿反应,而一些研究已经发现Mn(II)和Cu(II)等金属离子也可以引发类芬顿反应。然而,Fe(II)在封装和运载过程容易氧化形成Fe(III),并且,在Fe(II)介导的芬顿反应中,Fe(II)催化H2O2生成·OH,其自身可以氧化生成Fe(III)。Fe(II)的催化性能高于Fe(III),将Fe(III)转化为Fe(II)的效率将影响芬顿反应的效果。因此,用还原性物质加快Fe(III)还原为Fe(II)的速率可能更好地实现CDT。基于多酚类物质与Fe(III)之间的配位相互作用形成超微小金属多酚网络(GA/Fe(III))纳米复合物,可以作为芬顿反应催化剂,将胞内H2O2转化为强氧化的·OH,显著提高CDT效率。目前的多酚类物质与Fe(III)的纳米复合物只是将多酚物质与Fe(III)盐简单混合形成复合物,得到的纳米颗粒粒径不具有可控性并且粒径过大,在体内穿过率低,无法进行长循环。
药物载体对肿瘤局部微环境有特异性的反应会避免对正常组织造成损伤。与正常细胞相比,肿瘤细胞中pH更低,这为癌症特异性治疗提供了一个靶点。金属有机框架(Metal-organic frameworks,MOF)是通过中心金属离子或金属团簇与有机配体自组装形成的一类具有周期性结构的多孔材料。ZIF-8是由锌离子和2-甲基咪唑配位形成的一种新型多孔复合材料,具有孔隙率高、结构规整、表面功能可调、低pH响应性降解、良好的生物相容性等优点,广泛用于制备药物纳米载体。虽然构建刺激响应型纳米载体可以增加肿瘤特异性药物释放,但由于肿瘤的异质性、肿瘤微环境的复杂性和不稳定性,难以实现靶点位置精确控制药物释放,因此,现有的ZIF-8靶向性差,无法对肿瘤细胞进行特异性识别和摄取。
发明内容
针对现有技术的不足,本发明提供一种长循环多功能金属有机框架纳米制剂的制备方法。
本发明得到的多功能金属有机框架纳米制剂,可以同时实现肿瘤原位成像和治疗,由于聚乙二醇的掺杂可以在体内实现隐身,实现长循环,表面包覆透明质酸实现可以对肿瘤细胞进行特异性识别和摄取,具有良好的靶向性,封装基于聚乙烯吡咯烷酮GA/Fe纳米复合物使该制剂同时实现基于Fe3+的T2成像效果,可以准确获得肿瘤空间位置,没食子酸可还原芬顿反应产生的Fe3+,加速芬顿反应产生·OH有效杀死肿瘤细胞;在肿瘤治疗中具有良好的应用前景。
为解决以上问题,本发明是通过以下技术方案实现的:
一种长循环多功能金属有机框架纳米制剂的制备方法,包括步骤如下:
1)将没食子酸(GA)溶液在剧烈搅拌条件下滴入氯化铁(FeCl3)与矿化剂的混合液中,得混合液a,混合液a搅拌反应;反应后的溶液透析后冻干,制得GA/Fe纳米复合物(GA/FeNPs);
2)将聚乙二醇、葡萄糖氧化酶(GOx)、阿霉素(Dox)、GA/Fe纳米复合物和2-甲基咪唑溶液混合均匀,然后加入硝酸锌溶液溶液,混合均匀,得混合液b,将混合液b搅拌反应;反应后的溶液离心分离,然后水洗、除菌,获得白色纳米颗粒;
3)将白色纳米颗粒溶解,浸泡于透明质酸溶液中进行孵育,然后离心分离,水洗,除菌,制得多功能金属有机框架纳米制剂。
根据本发明优选的,步骤1)中,矿化剂为聚乙烯吡咯烷酮,聚乙烯吡咯烷酮PVP分子量为10-150 kDa。
根据本发明优选的,步骤1)中,氯化铁(FeCl3)与矿化剂的混合液溶剂为水,没食子酸(GA)溶液的浓度为0.5-2mg/mL。
根据本发明优选的,步骤1)中,没食子酸(GA)与氯化铁(FeCl3)、矿化剂的摩尔比为1:(1-3):(3-7)。
最为优选的,步骤1)中,没食子酸(GA)与氯化铁(FeCl3)、矿化剂的摩尔比为1:2:5。
根据本发明优选的,步骤1)中,GA/Fe纳米复合物的粒径为10-40nm。
本发明采用聚乙烯吡咯烷酮为矿化剂,制备了粒径可控的GA/Fe纳米复合物,得到的GA/Fe纳米复合物的粒径为10-40nm,包封率高,粒径适中,避免了现有直接混合得到的复合物粒径较大,包封率低,甚至不能被包封的缺陷;同时实现了在T2通道进行核磁成像。
根据本发明优选的,步骤1)中,所述没食子酸为分析纯,氯化铁为六水合氯化铁。
根据本发明优选的,步骤2)中,所述聚乙二醇为矿化剂,聚乙二醇分子量为5-40kDa,为端基为羟基的八臂聚乙二醇。
根据本发明优选的,步骤2)中,聚乙二醇、葡萄糖氧化酶(GOx)、GA/Fe纳米复合物的质量比为(1-20):(1-5):(5-100)。
根据本发明优选的,步骤2)中,2-甲基咪唑溶液的浓度为20-340 mmol/L,进一步优选的,2-甲基咪唑溶液的浓度为160 mmol/L。
根据本发明优选的,步骤2)中,聚乙二醇与2-甲基咪唑溶液的质量体积比为(1-20):(1-10),单位:mg/mL。
根据本发明优选的,步骤2)中,葡萄糖氧化酶分子量为40-160 kDa,进一步优选的,葡萄糖氧化酶分子量为160 kDa。
根据本发明优选的,步骤2)中,葡萄糖氧化酶为默克美国sigma化学公司的葡萄糖氧化酶。
根据本发明优选的,步骤2)中,聚乙二醇与阿霉素(Dox)的质量比为(1-20):(0.5-3)。
根据本发明优选的,步骤2)中,硝酸锌溶液的浓度为10-60 mmol/L,进一步优选的,硝酸锌溶液的浓度为40 mmol/L。
根据本发明优选的,步骤2)中,阿霉素(Dox)与硝酸锌溶液的质量体积比为(0.5-3):(1-10),单位:mg/mL。
根据本发明优选的,步骤2)中,反应温度为25-60℃,反应时间为0.5-2小时,优选的,反应温度为25-30℃,反应时间为1小时。
根据本发明优选的,步骤2)中,所述溶液离心分离所用的离心力为2000-80000 g,优选的,溶液离心分离所用的离心力为5000-8000 g。
根据本发明优选的,步骤2)中,得到的白色颗粒粒径为150-800 nm,进一步优选的,白色颗粒的粒径为200-400 nm。
根据本发明优选的,步骤3)中,所述的透明质酸分子量为100-500 kDa,进一步优选的,透明质酸分子量为110 kD。
本发明透明质酸分子量使多功能金属有机框架纳米制剂靶向性好,可以对肿瘤细胞进行特异性识别和摄取,分子量小,靶向性差,随着分子量的增大,靶向性逐渐增强,但分子量过大,容易造成制剂难以溶解。
根据本发明优选的,步骤3)中,透明质酸溶液的浓度为1-2.8mg/mL,进一步优选的,透明质酸溶液的浓度为1 mg/mL。
根据本发明优选的,步骤3)中,孵育时间为1-5h。
透明质酸浓度过大、孵育时间过长时,尤其是浓度大于3 mg/mL,孵育时间超过6h,得到的纳米颗粒会出现刻蚀现象。(如图3所示)
本发明得到多功能金属有机框架纳米制剂颗粒表面光滑,性能稳定,药物的生物利用度高。
根据本发明优选的,步骤3)中,所述离心分离所用的离心力为2000-60000 g,优选的,离心分离所用的离心力为2000-6000 g。
根据本发明优选的,步骤3)中,得到的纳米药物制剂中纳米粒子粒径为200-800nm,进一步优选的,纳米药物制剂中纳米粒子的粒径为250-450 nm。
本发明的技术特点及优点如下:
1、本发明得到的多功能金属有机框架纳米制剂,可以同时实现肿瘤原位成像和治疗,在体内隐身性好,可以实现长循环,并且靶向性好,可以对肿瘤细胞进行特异性识别和摄取,实现基于Fe3+的T2成像效果,可以准确获得肿瘤空间位置,没食子酸可还原芬顿反应产生的Fe3+,加速芬顿反应产生·OH有效杀死肿瘤细胞;具有优异的生物相容性、肿瘤靶向能力、双模态成像功能和联合化学动力学,饥饿治疗和化疗的能力,解决了多药递送所面临的药物装载问题。
2、本发明采用聚乙烯吡咯烷酮为矿化剂,制备了粒径可控的GA/Fe纳米复合物,得到的GA/Fe纳米复合物的粒径为10-40nm,包封率高,粒径适中,避免了现有直接混合得到的复合物粒径较大,避免了现有直接混合得到的复合物粒径较大,包封率低,甚至不能被包封的缺陷;同时实现了在T2通道进行核磁成像。
3、本发明的金属有机框架纳米药物制剂,直接在反应过程中加入一线抗肿瘤药物及生物活性蛋白一锅法制得,装载过程中不涉及有机试剂、微波、高温等剧烈条件,不会对药物和活性蛋白的结构和活性造成破坏,并且制备工艺简单、成熟稳定,可以大规模生产。
附图说明
图1为实施例1制得的 GA/Fe(III)纳米复合物的TEM图像,标尺为200 nm;
图2为实施例1、对比例1-5制得的封装不同药物的纳米颗粒扫描电镜照片,a1为对比例3的透射电镜图,b1为对比例2的扫描电镜图,c1为对比例1的扫描电镜图,d1为对比例4的扫描电镜图,e1为实施例1的扫描电镜图,标尺均为200 nm。;
图3为对比例5纳米颗粒的扫描电镜照片。
图4为实施例1、对比例4与4T1细胞共孵育8小时后的共聚焦图像;
图5为实施例1、对比例1-5与4T1细胞孵育24 h后的细胞存活率示意图。
图6为实施例1、对比例1-5尾静脉注射治疗后14天小鼠肿瘤体积变化图;
图7为实施例1、对比例4不同浓度下和不同时间点的体外T2 MRI成像;
图8为尾静脉注射实施例1和对比例4不同时间点在各器官(2 h、4 h、8 h、12 h和24 h)的生物分布图。
具体实施方式:
为更好地理解本发明,下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件,按照常规条件进行。所用试剂均为可以通过市场购买获得的常规产品。
实施例1:
长循环多功能金属有机框架纳米制剂的制备方法,具体如下:
(1)将没食子酸(GA)溶液在剧烈搅拌条件下滴入氯化铁(FeCl3)与聚乙烯吡咯烷酮(PVP)的混合液中搅拌反应12 h;没食子酸(GA)与氯化铁(FeCl3)、矿化剂的摩尔比为1:2:5;聚乙烯吡咯烷酮(PVP)的分子量为40kD;
(2)反应结束后将溶液透析后冻干,制得GA/Fe纳米复合物;GA/Fe(III)纳米复合物的TEM图见图1所示,通过图1可以看出,GA/Fe(III)纳米复合物的粒径均匀,平均粒径在20nm;
(3)将10 mg分子量为40 kDa的八臂聚乙二醇(8-arm-PEG-OH)、1 mg葡萄糖氧化酶(GOx)、1 mg阿霉素(Dox)、50 mg GA/Fe纳米复合物和2 mL的2-甲基咪唑(2-MIM,160 mM)混合均匀,然后加入2 mL的硝酸锌溶液(Zn(NO3)2,40 mM),常温下搅拌反应1 h;
(4)反应结束后将混合溶液离心(7000 g,3 min),反复操作4次,每次离心后补加4mL超纯水,以除去未反应的物质,除菌后离心。
(5)将离心获得的纳米颗粒超声分散于透明质酸溶液中反应3 h,透明质酸浓度为1mg/mL,透明质酸分子量为110 kD;然后离心分离,水洗,除菌,制得靶向纳米制剂;记作Dox&GGF@ZIF-8@HA。
对比例1:
同实施例1所述的纳米药物制剂的制备方法,不同之处在于:
未加入葡萄糖氧化酶(GOx),记作GGF@ZIF-8。
对比例2:
同实施例1所述的疫苗制剂的制备方法,不同之处在于:
未加入葡萄糖氧化酶和GA/Fe纳米复合物,记作Dox@ZIF-8。
对比例3:
同实施例1所述的疫苗制剂的制备方法,不同之处在于:
未加入阿霉素(Dox)、葡萄糖氧化酶(GOx)和GA/Fe纳米复合物,记作ZIF-8。
对比例4:
同实施例1所述的疫苗制剂的制备方法,不同之处在于:
未与透明质酸溶液共孵育;记作Dox&GGF@ZIF-8。
对比例5:
同实施例1所述的疫苗制剂的制备方法,不同之处在于:
透明质酸溶液浓度为3 mg/mL。
应用实验例:
实验例1:4T1细胞对纳米药物制剂的摄取
培养至对数期的4T1细胞(密度80~90%),用0.25%胰酶消化,每孔5×103个细胞的密度接种于四格共聚焦皿中,培养箱中培养24 h至细胞贴壁(37 °C、5% CO2)。吸去培养基,加入PBS清洗24孔培养板3次,然后培养过夜, 4T1细胞用含有31.2 mg/mL Dox&GGF@ZIF-8(对比例4)和Dox&GGF@ZIF-8@HA(实施例1)的培养基置换旧培养基,继续培养8小时后,多聚甲醛固定细胞, Hoechst 33342染核,WGA633染膜后吸去染液,加入防荧光淬灭剂保存,使用共聚焦显微镜观察样品。结果如图4所示,通过图4可以看出,实施例1的药物纳米制剂可以特异性识别的肿瘤细胞,更容易被细胞吞噬,靶向性强。
实验例2:纳米药物制剂对4T1细胞增殖影响
培养4T1细胞生长至对数期,消化、收集加入至96孔细胞培养板中(3×104cells/mL),培养24 h至细胞贴壁。分别加入不同浓度的ZIF-8(对比例3)、Dox@ZIF-8(对比例2)、GGF@ZIF-8(对比例1)、Dox&GGF@ZIF-8(对比例4)和Dox&GGF@ZIF-8@HA(实施例1),PBS作为空白对照,培养箱中继续培养24 h。取出96孔板,无菌条件下每孔加入20 μL MTT溶液(5mg/mL),继续孵育4 h。弃去上清后,每孔中加入150 μL DMSO溶解甲瓒颗粒。完全溶解后,用酶标仪检测570 nm处吸光度值(OD570)。细胞活力测定结果如图5所示,结果显示,本发明金属有机框架药物制剂具有良好的抑制肿瘤细胞增殖的效果。
实验例3:纳米药物制剂体内抗肿瘤效果效果测定
通过在雌性Balb/ca小鼠右下肢皮下注射100 μL浓度为1 × 108/mL4T1细胞溶液并养殖,待肿瘤体积增长至100 mm3时开始治疗。
将4T1荷瘤小鼠自由分为6组,每组6只小鼠。每组小鼠分别尾静脉注射200 μLPBS、ZIF-8(对比例3)、Dox@ZIF-8(对比例2)、GGF@ZIF-8(对比例1)、Dox&GGF@ZIF-8(对比例4)和Dox&GGF@ZIF-8@HA(实施例1),为了测试这些纳米颗粒的治疗效果,治疗过程中监测这些小鼠的肿瘤大小和体重。测试结果如6所示,通过图6对比,可以看出,Dox&GGF@ZIF-8@HA(实施例1)肿瘤抑制效果最优。
实验例4:纳米药物制剂体内荧光成像和核磁成像效果测定
通过在雌性Balb/ca小鼠右下肢皮下注射100 μL浓度为1 × 108/mL4T1细胞溶液并养殖,待肿瘤体积增长至100 mm3时尾静脉注射100 μL Dox&GGF@ZIF-8(对比例4)和Dox&GGF@ZIF-8@HA(实施例1)。并在注射后的不同时间用小动物成像仪及核磁成像对小鼠进行荧光和核磁成像。
不同浓度下和不同时间点的体外T2 MRI成像见图7所示,尾静脉注射实施例1和对比例4不同时间点在各器官的生物分布图见图8所示。
综上:本发明金属有机框架药物制剂在全身给药4 h后,可以利用靶向和EPR效应在肿瘤部位特异性富集。并且具有良好的体内核磁成像效果。基于以上实验结果,我们可以得出结论,Dox&GGF@ZIF-8@HA可以作为一种有前途的药物用于T2加权MRI显影和荧光多模式成像引导的抗肿瘤治疗。
实施例2:
同实施例1所述长循环多功能金属有机框架纳米制剂的制备方法,不同之处在于:
步骤(1)中,聚乙烯吡咯烷酮(PVP)的分子量为60kD。
实施例3:
同实施例1所述长循环多功能金属有机框架纳米制剂的制备方法,不同之处在于:
步骤(1)中,聚乙烯吡咯烷酮(PVP)的分子量为80kD。
实施例4:
同实施例1所述长循环多功能金属有机框架纳米制剂的制备方法,不同之处在于:
步骤(1)中,没食子酸(GA)与氯化铁(FeCl3)、矿化剂的摩尔比为1:3:7,其他按实施例1进行。
实施例5:
同实施例1所述长循环多功能金属有机框架纳米制剂的制备方法,不同之处在于:
步骤(1)中,没食子酸(GA)与氯化铁(FeCl3)、矿化剂的摩尔比为1:1:3,其他按实施例1进行。
实施例6:
同实施例1所述长循环多功能金属有机框架纳米制剂的制备方法,不同之处在于:
步骤(5)中,将离心获得的纳米颗粒超声分散于透明质酸溶液中反应2 h,透明质酸浓度为2mg/mL,透明质酸分子量为110 kD;然后离心分离,水洗,除菌,制得靶向纳米制剂。
实施例7:
同实施例1所述长循环多功能金属有机框架纳米制剂的制备方法,不同之处在于:
步骤(5)中,将离心获得的纳米颗粒超声分散于透明质酸溶液中反应3h,透明质酸浓度为1.5mg/mL,透明质酸分子量为220 kD;然后离心分离,水洗,除菌,制得靶向纳米制剂。
当然,本发明还可以有多种实施例,在不背离本发明精神及其实质的情况下,熟悉本领域的技术人员可根据本发明的公开做出各种相应的改变和变形,但这些相应的改变和变形都应属于本发明的权利要求的保护范围。
Claims (8)
1.一种长循环多功能金属有机框架纳米制剂的制备方法,包括步骤如下:
1)将没食子酸(GA)溶液在剧烈搅拌条件下滴入氯化铁(FeCl3)与矿化剂的混合液中,得混合液a,混合液a搅拌反应;反应后的溶液透析后冻干,制得GA/Fe纳米复合物;矿化剂为聚乙烯吡咯烷酮,聚乙烯吡咯烷酮PVP分子量为10-150 kDa;氯化铁(FeCl3)与矿化剂的混合液溶剂为水,没食子酸(GA)溶液的浓度为0.5-2mg/mL;没食子酸(GA)与氯化铁(FeCl3)、矿化剂的摩尔比为1:(1-3):(3-7);
2)将聚乙二醇、葡萄糖氧化酶、阿霉素、GA/Fe纳米复合物和2-甲基咪唑溶液混合均匀,然后加入硝酸锌溶液溶液,混合均匀,得混合液b,将混合液b搅拌反应;反应后的溶液离心分离,然后水洗、除菌,获得白色纳米颗粒;
聚乙二醇为矿化剂,聚乙二醇分子量为5-40 kDa,为端基为羟基的八臂聚乙二醇;聚乙二醇、葡萄糖氧化酶、GA/Fe纳米复合物的质量比为(1-20):(1-5):(5-100);
2-甲基咪唑溶液的浓度为20-340 mmol/L,聚乙二醇与2-甲基咪唑溶液的质量体积比为(1-20):(1-10),单位:mg/mL;
葡萄糖氧化酶分子量为40-160 kDa,聚乙二醇与阿霉素的质量比为(1-20):(0.5-3);硝酸锌溶液的浓度为10-60 mmol/L,阿霉素与硝酸锌溶液的质量体积比为(0.5-3):(1-10),单位:mg/mL;
3)将白色纳米颗粒溶解,浸泡于透明质酸溶液中进行孵育,然后离心分离,水洗,除菌,制得多功能金属有机框架纳米制剂;
所述的透明质酸分子量为100-500 kDa;
透明质酸溶液的浓度为1-2.8mg/mL,孵育时间为1-5h。
2.根据权利要求1所述的制备方法,其特征在于,步骤1)中,没食子酸(GA)与氯化铁(FeCl3)、矿化剂的摩尔比为1:2:5;GA/Fe纳米复合物的粒径为10-40nm;所述没食子酸为分析纯,氯化铁为六水合氯化铁。
3.根据权利要求1所述的制备方法,其特征在于, 步骤2)中, 2-甲基咪唑溶液的浓度为160 mmol/L。
4.根据权利要求1所述的制备方法,其特征在于,步骤2)中,葡萄糖氧化酶分子量为160kDa;硝酸锌溶液的浓度为40 mmol/L。
5.根据权利要求1所述的制备方法,其特征在于,步骤2)中,反应温度为25-60℃,反应时间为0.5-2小时;所述溶液离心分离所用的离心力为2000-80000 g;得到的白色纳米颗粒粒径为150-800 nm。
6.根据权利要求1所述的制备方法,其特征在于,步骤3)中,所述的透明质酸分子量为110 kD。
7.根据权利要求1所述的制备方法,其特征在于,步骤3)中,透明质酸溶液的浓度为1mg/mL。
8.根据权利要求1所述的制备方法,其特征在于,步骤3)中,所述离心分离所用的离心力为2000-60000 g。
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