CN116327627A - 一种四面体框架核酸-光甘草定复合物及其在皮肤美白方面的用途 - Google Patents
一种四面体框架核酸-光甘草定复合物及其在皮肤美白方面的用途 Download PDFInfo
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- CN116327627A CN116327627A CN202310423356.5A CN202310423356A CN116327627A CN 116327627 A CN116327627 A CN 116327627A CN 202310423356 A CN202310423356 A CN 202310423356A CN 116327627 A CN116327627 A CN 116327627A
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- Cosmetics (AREA)
Abstract
本发明提供了一种四面体框架核酸‑光甘草定复合物及其在皮肤美白方面的用途,属于生物医药技术领域。本发明四面体框架核酸‑光甘草定复合物是由四面体框架核酸和光甘草定混合后形成的复合物。本发明复合物以tFNA为载体进行光甘草定的协同递送,将光甘草定运送至靶点细胞附近,精准地抑制黑色素合成相关通路中的酶,有效地减少黑色素产生,达到皮肤美白的目的,机制清晰,效果明显。本发明复合物组织渗透性和生物相容性好,能够通过透皮给药途径,无创害地给药,并取得优异的效果,具有良好的应用前景。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种四面体框架核酸-光甘草定复合物及其在皮肤美白方面的用途。
背景技术
色素沉着是一种常见的皮肤疾病,类型包括黑斑病、雀斑、老年斑和炎症后色素沉着等多种类型。尽管每种类型的临床表现和病理表现有所不同,黑色素生成增加和分布不均是色素沉着过度的主要原因。在炎症反应中,活性氧和一些细胞因子可以刺激黑色素生成和分布,会导致色素沉着。此外,紫外线(UV)辐射也可以破坏皮肤细胞的DNA,激活黑色素细胞,进而导致色素沉着。目前针对色素沉着的治疗方法包括:①外用药物:氢醌是用来治疗色素沉着最主要的外用药物,它是一种酚类化合物,可通过抑制酪氨酸酶来阻羟基苯丙氨酸向黑色素的转化。但长期使用氢醌可能会导致接触性皮炎和色素沉着不足,而且氢醌的性质不稳定,其氧化副产物如醌等物质具有很强的黑素毒性,这使得目前氢醌的使用仍然存在争议;②口服药物:有研究表明口服氨甲环酸可通过抑制黑素细胞增殖及酪氨酸酶活性、抑制炎症反应及局部肥大细胞浸润来达到减少黑色素生成的目的,但临床证据不足,效果有限;③化学剥脱:化学剥脱是指用化学试剂对皮肤进行可控性的破坏,启动创伤修复,促进表皮重建的过程,常用的剥脱剂有乙醇酸、水杨酸等药物。但治疗过程控制不当可能对皮肤产生伤害,诱发新的色素沉着;④光电治疗:有研究报道使用调Q1 064nm掺钕钇铝石榴石激光、660nm LED等对色素沉着有治疗作用,但光电治疗在医学界仍有一定争议,目前缺乏大型随机临床试验,而且其治疗本身可能会加重色素沉着。可见上述治疗方法均存在一定问题。尚没有针对色素沉着的标准治疗方法,因此,针对黑色素生成和分布不均的机制,开发新的治疗方法是治疗色素沉着实现皮肤美白的重要研究方向。
光甘草定(Gla)是一种天然异黄酮,在抑制酪氨酸酶(黑色素合成中的关键酶)活性、清除自由基和抗氧化方面有着显著效果。因此,Gla在治疗皮肤增白、光老化和减轻色素沉着和红斑方面具有潜在的应用前景。然而Gla存在一些问题,如水溶性差、生物利用度低、缺乏有效的载体等,这些问题都会影响其在皮肤屏障中的渗透和作用效果。开发有效的载体可以使Gla更好地发挥其作用,对于提高Gla在皮肤疾病治疗中的应用价值具有重要意义。
四面体框架核酸(tFNA)是一种由四条特定序列的DNA单链通过一定步骤组装成的一种新型的生物纳米材料,因其结构稳定性、细胞渗透性和生物相容性,在药物递送、疾病治疗和生物医学成像等方面有着广阔的应用前景。tFNA的稳定四面体结构使其能够通过与细胞相互作用,顺利穿过细胞膜屏障,具有携带各类物质进入细胞的潜力。目前,tFNA已被证明可以递送小分子药物、多肽和寡核苷酸药物,依靠其在细胞渗透中的生物学和结构特性,tFNA可以采用较低的静电排斥力通过多孔蛋白内化进入细胞。tFNA作为一种具有广阔应用前景的生物纳米材料,为药物递送和疾病治疗提供了新的策略。
但是由于tFNA的结构特殊,且具有一定活性,因此其与小分子药物结合后两者相互作用如何,是发挥协同作用还是拮抗作用,这是未知的,不同小分子药物效果可能会出现较大的不同。目前尚未见tFNA负载光甘草定形成的复合物,tFNA能否成功结合光甘草定,使其发挥更好的效果需要进一步研究。
发明内容
本发明的目的是提供一种四面体框架核酸-光甘草定复合物及其在皮肤美白方面的用途。
本发明提供了一种四面体框架核酸-光甘草定复合物,它是由四面体框架核酸和光甘草定混合后形成的复合物。
进一步地,所述四面体框架核酸和光甘草定混合时,四面体框架核酸和光甘草定的摩尔比为1:20~1:320。
进一步地,所述四面体框架核酸和光甘草定的摩尔比为1:160~1:250;
优选地,所述四面体框架核酸和光甘草定的摩尔比为1:160。
进一步地,所述四面体框架核酸由四条DNA单链自组装合成;所述四条DNA单链的序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、和SEQ ID NO.4所示。
进一步地,所述四面体框架核酸的合成方法包括以下步骤:将四条DNA单链加入到TM缓冲液中,95℃维持10min,4℃维持20min以上,即得。
进一步地,所述四条DNA单链为等摩尔比的四条DNA单链。
本发明还提供了一种制备前述的四面体框架核酸-光甘草定复合物的方法,它包括如下步骤:
将四面体框架核酸和光甘草定加入溶剂中混合反应,即得;
优选地,所述溶剂为PBS、DMSO中的一种或多种;
和/或,所述四面体框架核酸的浓度为200~250nM;
和/或,所述反应的温度为20~30℃;
和/或,所述反应的时间为4~6h。
本发明还提供了前述的四面体框架核酸-光甘草定复合物在制备具有美白功效的药物或化妆品中的用途;
优选地,所述药物或化妆品是减少皮肤色素沉着、恢复皮肤白皙和/或均匀肤色的药物或化妆品;
更优选地,所述药物或化妆品是抑制黑色素生成的药物或化妆品。
进一步地,所述药物或化妆品是抑制酶TYR、MITF、TRP-1和/或TRP-2表达的药物或化妆品。
进一步地,所述药物为通过透皮给药的药物。
与现有技术相比,本发明的有益效果为:
本发明提供了一种四面体框架核酸-光甘草定复合物,其以tFNA为载体进行光甘草定的协同递送,将光甘草定运送至靶点细胞附近,精准地抑制黑色素合成相关通路中的酶,有效地减少黑色素产生,达到皮肤美白的目的,机制清晰,效果明显。本发明复合物组织渗透性和生物相容性好,能够通过透皮给药途径,无创害地给药,并取得优异的效果,具有良好的应用前景。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为tFNA-Gla复合物的合成方法和表征结果:A为tFNA-Gla复合物的具体合成过程;B为PAGE聚丙烯酰胺凝胶电泳结果;C为GelRed荧光强度结果;D为tFNA和tFNA-Gla复合物的粒径结果;E为tFNA和tFNA-Gla复合物的电位结果;F为tFNA和tFNA-Gla复合物的粒径、电位统计结果;G为tFNA-Gla复合物的TEM图像;H为tFNA-Gla复合物的AFM图像。
图2为细胞摄取实验检测结果和黑色素含量测定结果:A为使用tFNA-Gla复合物(tFNA:Gla摩尔比=1:160,tFNA浓度为250nM)培养不同时间的细胞荧光染色图片;B为流式细胞检测结果,图中红色为Ctrl,黄色为tFNA,蓝色为tFNA-Gla复合物;C为CCK-8细胞活性检测结果;D为黑色素含量测定结果。
图3为tFNA-Gla复合物对相关蛋白表达的影响:A为蛋白印迹法检测相关蛋白表达结果;B为蛋白印迹法检测结果的定量分析;C为TYR酪氨酸酶荧光染色结果;D为MITF小眼转录因子荧光染色结果。
图4为黄褐斑小鼠模型的建立以及治疗结果:A为黄褐斑小鼠模型的建模流程和给药方法;B为建模结果;C和D为治疗的宏观图;E为治疗统计结果。
图5为动物模型切片结果:A为小鼠耳朵涂抹Cy5荧光标记的tFNA-Gla后真皮层的荧光信号;B为各组表皮厚度统计结果;C为HE染色结果;D为Masson染色结果。
具体实施方式
本发明具体实施方式中使用的原料、设备均为已知产品,通过购买市售产品获得。
实施例1、四面体框架核酸-光甘草定(tFNA-Gla)复合物的制备
(1)四面体框架核酸的合成
在TM缓冲液(50mM MgCl2和10mM TrisHCl,pH 8.0)中加入等摩尔量的四种特殊序列DNA单链(使得溶解后四种DNA单链的终浓度均为1000nM),溶解后充分混合均匀,然后在一系列加热程序(95℃维持10min,迅速降温至4℃维持20min以上)后,自组装合成得到四面体框架核酸(tFNA)。四条DNA单链的具体序列如下表1所示。
表1.DNA单链的具体序列
(2)tFNA-Gla复合物的制备
将光甘草定(Gla)溶解于DMSO中,再与tFNA混合(混合后tFNA的浓度为250nM,tFNA与Gla的摩尔比为1:20、1:40、1:80、1:160或1:320),然后用磁力搅拌器持续避光搅拌6小时(20℃),Gla会通过嵌插结合搭载到tFNA上,形成tFNA-Gla复合物,具体合成过程示意图如图1A所示。
(3)tFNA-Gla复合物的鉴定
①通过PAGE聚丙烯酰胺凝胶电泳验证tFNA和tFNA-Gla复合物的合成,如图1B所示,泳道1为S1;泳道2为S1+S2;泳道3为S1+S2+S3;泳道4为tFNA;泳道5为tFNA:Gla=1:20;泳道6为tFNA:Gla=1:40;泳道7为tFNA:Gla=1:80;泳道8为tFNA:Gla=1:160;泳道9为tFNA:Gla=1:320。结果显示tFNA和tFNA-Gla的条带较为干净集中,表明了tFNA和tFNA-Gla的成功合成。
②通过对不同比例的tFNA-Gla复合物(tFNA:Gla摩尔比=1:20-320,tFNA浓度为250nM)进行GelRed染色,检测GelRed荧光强度验证tFNA-Gla复合物的合成,tFNA作为对照,结果如图1C所示,tFNA-Gla复合物的GelRed荧光强度明显低于tFNA的GelRed荧光强度,说明Gla竞争结合了GelRed的结合位点,表明Gla成功搭载到了tFNA上。
③通过动态光散射仪(DLS)测量tFNA和tFNA-Gla复合物(tFNA:Gla=1:160)的粒径和电位,如图1D、1E、1F所示,tFNA-Gla复合物的粒径为21.50nm,tFNA-Gla复合物的电位为-6.49mV,表明了tFNA和tFNA-Gla复合物的成功合成。
④通过透射电子显微镜(TEM)和原子力显微镜(AFM)测量tFNA-Gla复合物(tFNA:Gla=1:160)的形态,如图1G和1H所示,有类似四面体结构的团状物产生。
以下通过具体试验例证明本发明的有益效果。
实验例1、细胞摄取实验
小鼠黑色素瘤细胞(B16细胞)在细胞培养箱(5%CO2,37℃)中培养,培养基由DMEM高糖培养基、10%胎牛血清和1%青霉素-链霉素溶液组成。
tFNA和tFNA-Gla复合物按照实施例1所述方法制备。
Cy5荧光物质标记的tFNA和tFNA-Gla复合物制备方法为:使用Cy5荧光物质标记实施例1中的S1链,按照实施例1的制备方法制备Cy5荧光物质标记的tFNA以及不同tFNA和Gla摩尔比的tFNA-Gla复合物。
CCK8实验:
为了探究tFNA作为空载体是否对细胞产生了毒性,以及tFNA-Gla复合物是否在进入细胞后诱导了细胞凋亡,采用CCK8检测tFNA以及tFNA-Gla复合物(不同tFNA和Gla摩尔比制备)对B16细胞的毒性影响,以单纯细胞培养作为对照(Ctrl)。具体方法如下:
(1)以1×103个/孔的细胞密度于96孔板中接种细胞,培养过夜使之贴壁;
(2)次日,用100μL含相同浓度的tFNA或tFNA-Gla复合物的低血清培养基替换原有培养基,继续培养24h后进行CCK8检测。
(3)加入CCK8试剂,避光孵育1.5小时;
(4)酶标仪检测450nm波长下OD值;
(5)导出检测数据,进行细胞活性的计算。
荧光染色实验:
具体方法如下:
(1)以1×105个/孔的细胞密度于细胞爬片中接种B16细胞,培养24h后,将培养基更换为含Cy5荧光物质标记的tFNA-Gla复合物(Cy5-tFNA-Gla,tFNA:Gla摩尔比=1:160,tFNA浓度为250nM)。
(2)在培养2h、4h、6h、8h后分别收集样本细胞,使用FITC标记的鬼笔环肽和DAPI染色后,用共聚焦显微镜拍摄荧光图像。
流式细胞术检测:
以1×103个/孔的细胞密度于96孔板中接种B16细胞,培养24h后,将培养基更换为含Cy5荧光物质标记的tFNA或tFNA-Gla复合物(tFNA:Gla摩尔比=1:160,tFNA浓度为250nM),以单纯细胞培养作为对照(Ctrl)。继续培养24h后收集细胞使用流式细胞仪检测携带Cy5荧光物质的细胞与总细胞的比率。
结果:
如图2C所示,通过CCK-8试剂盒确定了tFNA:Gla摩尔比=1:160(tFNA浓度为250nM)制备的tFNA-Gla复合物最佳。此时复合物可以在不对细胞产生毒性的情况下获得更大的给药浓度,从而发挥更好的治疗效果。
B16细胞在培养基中培养24小时后,将培养基更换为含Cy5-tFNA-Gla复合物(tFNA:Gla摩尔比=1:160,tFNA浓度为250nM)的培养基,在培养2h、4h、6h、8h后分别收集样本细胞,使用鬼笔环肽和DAPI染色后,用共聚焦显微镜拍摄荧光图像,结果如图2A所示,说明6小时后tFNA-Gla入胞最多。
②流式细胞术:流式细胞术检测如图2B所示,说明tFNA-Gla复合物入胞最多。
以上实验结果证明了本发明tFNA-Gla复合物入胞效果良好。
实验例2、黑色素含量测定和相关蛋白表达检测
1、黑色素含量测定:
实验分组:
(1)对照组(Ctrl):细胞+DEME高糖培养基+ddH2O+100mJ/cm2紫外光造模;
(2)tFNA-Gla组:细胞+DEME高糖培养基+ddH2O+100mJ/cm2紫外光造模+按照实施例1所述方法制备的tFNA-Gla复合物(tFNA:Gla摩尔比=1:80、1:160、1:200,tFNA浓度为250nM)。
方法:
(1)本发明采用B16细胞,以1×103个/孔的细胞密度于96孔板中接种B16细胞(DEME高糖培养基),培养24h;
(2)tFNA-Gla组分别加入等体积tFNA-Gla复合物(250nM,ddH2O作为溶剂)对B16细胞进行预处理,同时对细胞进行100mJ/cm2紫外光照射;对照组加入等体积ddH2O,同样使用紫外光照射(100mJ/cm2);
(3)处理12小时和24小时后,收集样本细胞并用NaOH溶解细胞内的黑色素。以溶液在475nm处的吸光度来代表黑色素含量。
结果如图2D所示,tFNA-Gla复合物显著减少了黑色素含量。
2、相关蛋白表达检测:
实验分组:
(1)空白组(Blank):细胞+DEME高糖培养基+ddH2O;
(2)对照组(Ctrl):细胞+DEME高糖培养基+ddH2O+100mJ/cm2紫外光造模;
(3)tFNA组:细胞+DEME高糖培养基+ddH2O+100mJ/cm2紫外光造模+按照实施例1所述方法制备的tFNA;
(4)Gla组:细胞+DEME高糖培养基+ddH2O+100mJ/cm2紫外光造模+Gla;
(5)tFNA-Gla组:细胞+DEME高糖培养基+ddH2O+100mJ/cm2紫外光造模+按照实施例1所述方法制备的tFNA-Gla复合物(tFNA:Gla摩尔比=1:160,tFNA浓度为250nM)。
方法:
TYR、MITF、TRP-1和TRP-2是黑色素合成过程中的关键酶。本发明采用B16细胞,按“黑色素含量测定”中的方法培养细胞24小时后,tFNA组、Gla组和tFNA-Gla组分别加入2mLtFNA(250nM)、Gla(50μM)和tFNA-Gla复合物(250nM)对B16细胞进行预处理,同时对细胞进行100mJ/cm2紫外光照射;对照组加入等体积ddH2O,使用紫外光照射(100mJ/cm2);空白组只加入等体积ddH2O,不使用紫外光照射。处理72小时后,提取样本中总蛋白质,通过蛋白印迹法检测相关蛋白表达。考虑到β-actin在B16细胞中的稳定表达,将其用作本实验的内参蛋白。
结果如图3A所示,与空白组相比,照射紫外光后,TYR、MITF、TRP-1和TRP-2的表达含量增加,tFNA-Gla组对应的条带清晰度降低,表明黑色素相关蛋白含量显著减少,图3B的定量分析反映了相同的结果。
按照上述方法处理B16细胞,然后进行荧光染色并使用共聚焦显微镜拍摄,观察TYR酪氨酸酶和MITF小眼转录因子的含量。结果如图3C和3D所示,tFNA-Gla组与黑色素合成相关的蛋白红色荧光明显减弱,表明相关蛋白含量减少。与单独的tFNA和Gla相比,发挥了协同增效的有益效果。
以上实验均证明了tFNA-Gla能够减少黑色素含量,抑制黑色素合成相关蛋白表达,且发挥了协同增效的效果。
实验例3、动物实验
1、建立动物模型
选择C57BL/6J小鼠(雌性,20g,8周),在SPF级环境下进行饲养,自由摄食。通过UVB照射(100mJ/cm2/d,两日一次,4周)小鼠,构建黄褐斑小鼠模型。
2、分组
随机分为空白组、对照组、Gla组、tFNA组、tFNA-Gla组和氢醌组:
(1)空白组:无特殊条件饲养,涂抹生理盐水+凡士林软膏;
(2)对照组:UVB照射100mJ/cm2/d,两日一次,4周;无特殊条件饲养,涂抹生理盐水+凡士林软膏;
(3)Gla组:UVB照射100mJ/cm2/d,两日一次,4周;无特殊条件饲养,涂抹40μM Gla+凡士林软膏;
(4)tFNA组:UVB照射100mJ/cm2/d,两日一次,4周;无特殊条件饲养,涂抹250nMtFNA+凡士林软膏;
(5)tFNA-Gla组:UVB照射100mJ/cm2/d,两日一次,4周;无特殊条件饲养,涂tFNA-Gla复合物(tFNA 250nM,Gla 40μM)+凡士林软膏;
(6)氢醌组:UVB照射100mJ/cm2/d,两日一次,4周;无特殊条件饲养,涂成品4%氢醌+凡士林软膏。
3、方法
①建模:为了探索减少小鼠中tFNA-Gla色素积累的能力,本发明通过UVB照射(100mJ/cm2/d,隔日4周)构建了黄褐斑小鼠模型,建模流程如图4A所示,建模结果如图4B所示。
②给药方法:将每组对应的药物和空白乳膏(凡士林乳膏:商品名:aquaphor)按体积比1:1混合,每日1次均匀涂抹于小鼠耳朵表面,持续14天,并且每日用相机在相同拍摄环境、相同参数、相同角度拍摄小鼠耳朵照片。
③数据分析:使用Adobe Photoshop 2020进行颜色分析,自动检查并记录每只小鼠的耳朵平均RGB值进行统计学分析,结果如图4C、4D、4E所示,tFNA-Gla组小鼠耳朵颜色明显变浅,效果与临床常用的4%氢醌相近。
④切片分析:给小鼠耳朵涂抹与空白乳膏(凡士林乳膏:商品名:aquaphor)按照体积比1:1混合的Cy5荧光标记的tFNA-Gla复合物(S1链上标记Cy5荧光标记,按照实施例1所述方法制备,tFNA和Gla的摩尔比为1:160)后,小鼠耳朵组织学切片中显示真皮层有明显荧光信号,如图5A所示,这表明药物成功渗透进入真皮层。同时收集给药后的小鼠的耳朵,进行组织学切片并且完成HE和Masson染色,结果如图5B、5C、5D所示,tFNA-Gla明显降低了黑色素的含量,而且还减轻了小鼠耳朵的炎症。
以上的动物实验表明,tFNA-Gla能够经皮吸收,有效减少组织内黑色素含量,还能缓解炎症。
综上,本发明提供了一种四面体框架核酸-光甘草定复合物,其以tFNA为载体进行光甘草定的协同递送,将光甘草定运送至靶点细胞附近,精准地抑制黑色素合成相关通路中的酶,有效地减少黑色素产生,达到皮肤美白的目的,机制清晰,效果明显。本发明复合物组织渗透性和生物相容性好,能够通过透皮给药途径,无创害地给药,并取得优异的效果,具有良好的应用前景。
Claims (10)
1.一种四面体框架核酸-光甘草定复合物,其特征在于:它是由四面体框架核酸和光甘草定混合后形成的复合物。
2.根据权利要求1所述的四面体框架核酸-光甘草定复合物,其特征在于:所述四面体框架核酸和光甘草定混合时,四面体框架核酸和光甘草定的摩尔比为1:20~1:320。
3.根据权利要求2所述的四面体框架核酸-光甘草定复合物,其特征在于:所述四面体框架核酸和光甘草定的摩尔比为1:160~1:250;
优选地,所述四面体框架核酸和光甘草定的摩尔比为1:160。
4.根据权利要求1~3任一项所述的四面体框架核酸-光甘草定复合物,其特征在于:所述四面体框架核酸由四条DNA单链自组装合成;所述四条DNA单链的序列分别如SEQ IDNO.1、SEQ ID NO.2、SEQ ID NO.3、和SEQ ID NO.4所示。
5.根据权利要求4所述的四面体框架核酸-光甘草定复合物,其特征在于:所述四面体框架核酸的合成方法包括以下步骤:将四条DNA单链加入到TM缓冲液中,95℃维持10min,4℃维持20min以上,即得。
6.根据权利要求5所述的四面体框架核酸-光甘草定复合物,其特征在于:所述四条DNA单链为等摩尔比的四条DNA单链。
7.一种制备权利要求1~6任一项所述的四面体框架核酸-光甘草定复合物的方法,其特征在于:它包括如下步骤:
将四面体框架核酸和光甘草定加入溶剂中混合反应,即得;
优选地,所述溶剂为PBS、DMSO中的一种或多种;
和/或,所述四面体框架核酸的浓度为200~250nM;
和/或,所述反应的温度为20~30℃;
和/或,所述反应的时间为4~6h。
8.权利要求1~6任一项所述的四面体框架核酸-光甘草定复合物在制备具有美白功效的药物或化妆品中的用途;
优选地,所述药物或化妆品是减少皮肤色素沉着、恢复皮肤白皙和/或均匀肤色的药物或化妆品;
更优选地,所述药物或化妆品是抑制黑色素生成的药物或化妆品。
9.根据权利要求8所述的用途,其特征在于:所述药物或化妆品是抑制酶TYR、MITF、TRP-1和/或TRP-2表达的药物或化妆品。
10.根据权利要求9所述的用途,其特征在于:所述药物为通过透皮给药的药物。
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