CN113754793B - 一种苯硼酸枝接的壳寡糖衍生物及其制备方法和应用 - Google Patents
一种苯硼酸枝接的壳寡糖衍生物及其制备方法和应用 Download PDFInfo
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- CN113754793B CN113754793B CN202010504960.7A CN202010504960A CN113754793B CN 113754793 B CN113754793 B CN 113754793B CN 202010504960 A CN202010504960 A CN 202010504960A CN 113754793 B CN113754793 B CN 113754793B
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- chitosan oligosaccharide
- phenylboronic acid
- derivative
- sirna
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Abstract
本发明为一种苯硼酸枝接的壳寡糖衍生物及其制备方法和应用,属于纳米生物医药材料领域。本发明涉及苯硼酸枝接的壳寡糖衍生物的合成及其制备方法,该衍生物能够通过静电作用和化学结合双重作用来实现对带负电siRNA药物的包载和压缩,制备粒径更小、更稳定的纳米粒;并且合成的衍生物能够控制释放siRNA,实现酸性肿瘤细胞内的药物快速释放。本发明将苯硼酸作为实现增加纳米粒稳定性、响应性释放的功能分子,将其与壳寡糖分子连接,解决了壳寡糖对siRNA递送效率不高的问题,提供了一种新颖、高效、低毒的药物递送载体及其制备方法与应用。
Description
技术领域
本发明属于纳米生物医药材料领域,具体涉及一种对基因药物高包载、强压缩并响应性释放药物的功能性聚合物载体材料及其制备方法和应用。
背景技术
基因治疗是指将治疗性基因导入靶细胞,从分子水平修正引起疾病的基因异常或基因缺陷,从而达到治疗或改善的效果。在基因治疗中,小干扰RNA(siRNA)是一类长度为20~25bp的双螺旋RNA(dsRNA),能够在体内诱导RNA干扰效应,实现特定基因的沉默。RNA基因治疗因其具有特异性强、无基因突变、通过合理的序列设计可以靶向任何感兴趣的mRNA等优点,在临床治疗上有巨大的潜力。裸siRNA分子在体内血清环境中极易被核酸酶降解,且带负电不易被细胞吸收,因此,siRNA必须借助特定的递送方法才能进入细胞。
壳聚糖具有生物相容性、可生物降解、安全性等优点,但水溶性差,只溶解于pH低于6.5的酸溶液中,且基本不溶于常用的有机试剂如二甲基甲酰胺、二甲基亚砜,限制了其应用和发展。壳寡糖是壳聚糖的低聚物,分子量一般小于10kDa,在很宽pH范围内水溶液中溶解性好,在大多数有机试剂中也有良好的溶解性。壳寡糖在溶液中带正电荷,通过静电作用能将带负电的siRNA包载或吸附在载体中,从而保护siRNA免受核酸酶的降解,还能提高细胞对siRNA的摄取。但壳寡糖在递送siRNA时存在不足:由于其分子量小,形成的纳米复合物稳定性相对较差,这可以通过对壳寡糖进行化学修饰来解决。
苯硼酸(PBA)是一种路易斯弱酸,能够与带顺式二醇结构的siRNA反应,并且形成的共价键是可逆的,pH>pKa时,PBA可以与siRNA通过共价键结合;当pH<pKa时,此反应在氢离子的作用下逆向进行,生成的共价键会断裂[1-3]。因此,将苯硼酸枝接到壳寡糖聚合物载体上,可以实现以下优势:(1)在形成纳米粒时,苯硼酸枝接的壳寡糖衍生物可以通过双重作用实现对siRNA包载和压缩,即:静电作用吸附带负电的siRNA、与siRNA的游离核糖结合形成复合物。因此,苯硼酸枝接的壳寡糖衍生物能实现对siRNA更强的包载、继而稳定性更好的纳米粒;(2)因PBA与siRNA的结合是可逆的,在纳米粒到达酸性的肿瘤环境时,PBA与siRNA之间的共价键逐渐断裂,因此纳米粒能够实现逐渐释放siRNA药物,实现肿瘤部位的响应性释放功能。
[1]Bin Yang,Huizhen Jia,Xuli Wang,Si Chen,Xianzheng Zhang,Renxi Zhuo,Jun Feng.Self-Assembled Vehicle Construction via Boronic Acid Coupling andHost-Guest Interaction for Serum-Tolerant DNA Transport and pH-ResponsiveDrug Delivery.Adv Healthc Mater.2014,3:596-608.
[2]Naito M,Yoshinaga N,Ishii T,Matsumoto A,Miyahara Y,Miyata K,Kataoka K.Enhanced Intracellular Delivery of siRNA by Controlling ATP-Responsivity of Phenylboronic Acid-Functionalized Polyion ComplexMicelles.Macromol Biosci.2018 Jan;18(1).
[3]Fan B,Kang L,Chen L,Sun P,Jin M,Wang Q,Bae YH,Huang W,GaoZ.Systemic siRNA Delivery with a Dual pH-Responsive and Tumor-targetedNanovector for Inhibiting Tumor Growth and Spontaneous Metastasis inOrthotopic Murine Model of Breast Carcinoma.Theranostic.2017,7:357-376.
发明内容
本发明要解决的问题的在于将苯硼酸作为实现增加纳米粒稳定性、响应性释放的功能分子,将其与低分子量的壳寡糖材料连接,提供了一种新的、高疗效、低毒性的多功能载体及其制备方法与应用。
本发明可以通过以下技术方案来实现:
本发明提供了一种苯硼酸枝接的壳寡糖衍生物,该衍生物由苯硼酸与壳寡糖分子链上的伯胺基通过化学键连接而形成。单体之间通过β-1,4糖苷键连接,壳寡糖的分子量为800~10000Da,优选为1000~8000Da,进一步优选为1500~6000Da,更优选为2000~4000Da,最优选为3000Da。与苯硼酸连接的仲胺基的摩尔数占壳寡糖中原有伯胺基总摩尔数的0~100%,优选为5~80%,进一步优选为10~50%,更优选为15%~40%,进一步更有选为19.1%。
本发明制备中选用2-溴甲基苯硼酸,也可用3-溴甲基苯硼酸、4-溴甲基苯硼酸替代,反应条件与使用2-溴甲基苯硼酸相同。2-溴甲基苯硼酸、3-溴甲基苯硼酸、4-溴甲基苯硼酸结构如下:
苯硼酸枝接的壳寡糖衍生物其制备方法为亲核取代反应,将苯硼酸与壳寡糖在有机试剂中溶解,在加热的条件下,壳寡糖上伯胺能够取代苯硼酸上的溴原子,苯硼酸最终通过C-N键与壳寡糖连接,生成苯硼酸枝接的壳寡糖衍生物。待反应结束,先后通过旋转蒸发法、真空干燥法除去有机试剂,选用透析法纯化反应物,最终通过冷冻干燥法得到最终纯化产物。优选的制备方法如下:
(1)将24.35mg苯硼酸、100mg壳寡糖在无水甲醇中溶解,70℃下加热回流,磁力搅拌反应24h。
(2)将反应物通过旋转蒸发仪法、真空干燥法除去甲醇。
(3)将反应物溶于双蒸水,选用截留分子量为200-3000的透析袋进行透析,优选截留分子量为500的透析袋:于去离子水中透析24h。
(4)在温度为-50℃、0.07Pa条件下,液体冷冻干燥24h后得冻干粉备用,得到苯硼酸枝接的壳寡糖衍生物(PBA-COS)。
步骤中所述的壳寡糖COS与苯硼酸摩尔比为1:3.4~1:13.6,优选1:3.4~1:8.5,进一步优选为1:3.4~1:5.1,最优选为1:3.4。
本发明将壳寡糖聚合物与苯硼酸相结合,制备了能够稳定纳米粒、控制药物释放的载体材料,可以直接或者再经过其他途径用于药物载体、基因载体等用途。
本发明的再一个方面,提供了上述的苯硼酸枝接的壳寡糖衍生物在作为体内、体外递送载体的应用。
在一个实施方案中,提供了聚合物纳米粒中包载基因药物为靶向survivin蛋白的siRNA。
包载siRNA的纳米粒制备方法参照康林等人的文献(参见Kang L,Fan B,Sun P,Huang W,Jin M,Wang Q,Gao Z.An effective tumor-targeting strategy utilizinghypoxia-sensitive siRNA delivery system for improved anti-tumor outcome.ActaBiomater.2016,44:341-54),进行粒径、透射电镜、基因递送效率的测定。
本发明提供的苯硼酸枝接的壳寡糖衍生物作为体外递送载体的应用,其方法和步骤如下:
(1)细胞增殖抑制
将材料冻干制剂用培养液稀释成预定浓度的溶液。每个浓度设6个复孔,并设阴性对照组和空白对照组。将适量对数生长期的细胞接种于96孔板中,继续培养24h后,介质以含有不同药物浓度的新鲜培养液替换,分别继续培养48h。每孔加入CCK-8试剂20μl。继续孵育2h,于450nm处测定吸收值,并以650nm为参考波长。计算细胞活力,公式如下:
细胞活力(%)=[(OD实验-OD空白对照)/(OD阴性对照-OD空白对照)]×100
(2)细胞摄取效率评价
以Cy5为荧光探针,连接到siRNA上。将适量对数生长期的B16F10细胞接种于24孔板中,继续培养24h后,介质更换为不含血清的培养液进行转染,将提前制备好的纳米粒溶液加入每孔,使每孔siRNA浓度为100nM。设置阴性对照组、裸siRNA组、未枝接的壳寡糖纳米粒组和苯硼酸枝接的壳寡糖纳米粒组,阳离子脂质体2000脂质复合物组作为阳性对照组,每组设置3个复孔。转染4h后,将细胞用PBS冲洗三遍,胰酶消化收集细胞并重悬至0.5mlPBS中,通过流式细胞仪检测细胞对纳米粒摄取效率。
(3)基因递送效率评价
将适量对数生长期的4T1luc细胞接种于24孔板中,继续培养24h后,介质更换为不含血清的培养液进行转染,将提前制备好的纳米粒溶液加入每孔,使每孔siRNA浓度为100nM。设置阴性对照组、裸siRNA组、未枝接的壳寡糖纳米粒组和苯硼酸枝接的壳寡糖纳米粒组,阳离子脂质体2000脂质复合物组作为阳性对照组,每组设置3个复孔。转染4h后,介质更换为含血清的新鲜培养液继续培养44h。之后将细胞用PBS冲洗,细胞裂解液裂解细胞收集RNA,之后用荧光素酶试剂盒检测荧光素酶基因的表达效率。
本发明提供的苯硼酸枝接的壳寡糖衍生物作为体内递送载体的应用,其方法和步骤如下:
(1)取4~6周龄的C57BL/6小鼠,建立小鼠体内黑色素瘤模型,待肿瘤体积长到70mm3,将小鼠随机分为5组,每组5只,5组分别瘤内注生理盐水、裸siRNA溶液、100μl的无苯硼酸枝接的壳寡糖纳米粒溶液和100μl的苯硼酸枝接的壳寡糖纳米溶液。
(2)以0.3mg siRNA/kg小鼠体重的剂量给药,每两天一次,共计给药四次。每两天检测小鼠的体重和肿瘤体积变化,直到最后一次给药后2天,脱颈处死全部小鼠,解剖肿瘤组织并称重;解剖肺、肝组织于中性甲醇中固定并H&E方法检测肿瘤转移。
有益效果:
1、合成过程中使用了一步亲核取代的合成方法,将苯硼酸通过取代反应连接到壳寡糖载体上,所采用分子量在800~9000Da的壳寡糖水溶性更好,比现有技术所采用的酰胺反应相比,取代反应简化了操作步骤,提高了工作效率,节约了生产成本。
2、本发明制备的纳米材料具有通过静电作用和化学结合双重作用来制备物理性质好、稳定的纳米粒的效果,最终制备的纳米粒粒径在98nm,远小于现有技术所报道的200~300nm左右,粒径更小的纳米粒在体内更稳定。
3、本发明制备纳米材料具有良好的生物相容性和控制药物释放的功能,从而能有效提高其递送和治疗的效果。
4、可选用苯硼酸枝接的壳寡糖衍生物作为为载体包载药物(如siRNA、阿霉素等带负电的模式药物),将其用本发明所制备的苯硼酸枝接的壳寡糖衍生物递送后,具有更好的纳米粒稳定性、更强的基因递送效率及抗肿瘤作用。
附图说明
图1:苯硼酸的1H-NMR图谱
图2:壳寡糖的1H-NMR图谱
图3:PBA-COS的1H-NMR图谱
图4:PBA-COS的FITR图谱
图5:包载siRNA的纳米粒的动态光散射(DLS)粒径分布图
图6:包载siRNA的纳米粒的透射电镜图
图7:纳米粒的在不同pH下的粒径分布图
图8:PBA-COS/siRNA纳米粒体外细胞摄取效率结果图
图9:PBA-COS/siRNA纳米粒体外基因沉默效率结果图
图10:PBA-COS和PBA-COS/siRNA纳米粒体外对小鼠黑色素瘤B16F10细胞的增殖抑制结果图
图11:PBA-COS/siRNA纳米粒体内抗小鼠黑色素瘤增殖结果图
图12:PBA-COS/siRNA纳米粒体内抗小鼠黑色素瘤肺转移结果图
具体实施方式
实施例1:苯硼酸枝接的壳寡糖衍生物的合成
称取100mg分子量为3000Da的壳寡糖溶于无水甲醇中,分别称取24.35mg、60.85mg和97.36mg的2-溴甲基苯硼酸溶于无水甲醇中,70℃加热回流,磁力搅拌反应24h。在加热的条件下,壳寡糖上伯胺能够取代苯硼酸上的溴原子,苯硼酸最终通过C-N键与壳寡糖连接,生成苯硼酸枝接的壳寡糖衍生物,分别制备了三种不同取代度的苯硼酸枝接的壳寡糖衍生物。
反应结束后,旋转蒸发仪、真空干燥法除去甲醇,随后溶于双蒸水选用截留分子量为500的透析袋于去离子水中透析24h。液体冷冻干燥后得到苯硼酸枝接的壳寡糖衍生物分子,下文简写为PBA-COS。
取冻干后的PBA-COS于D2O,采用600MHz的核磁共振氢谱(1H-NMR)进行结构确认。通过与苯硼酸(图1)与壳寡糖(图2)氢谱对比可知,在图3苯硼酸枝接的壳寡糖衍生物氢谱中,化学位移2.8~4.1ppm归属于PBA-COS结构中的葡萄糖五元环上的质子峰;并且,因硼酸基团有吸电子性质,故化学位移7.1~8.0ppm归属于苯硼酸上苯环上的质子峰,相比于无取代基苯环上的氢信号略有下移。以上峰归属表明苯硼酸通过与氨基反应,成功生成PBA-COS。并且通过积分计算苯硼酸的实际取代度,总结为表1。
表1不同壳寡糖衍生物实际取代度计算
红外光谱(图4)用于验证PBA-COS的结构,其中3200~3400cm-1的峰归属于壳寡糖分子中的游离羟基O-H伸缩振动,1601cm-1归属于苯硼酸苯环上的C=C双键伸缩振动,1337cm-1归属于苯硼酸上的B-O伸缩振动,进一步验证了核磁共振的结论。
实施例2:包载siRNA的PBA-COS/siRNA纳米粒的制备
选取取代度为19.1%的苯硼酸枝接的壳寡糖衍生物制备纳米粒,将siRNA干粉溶解于不含核酸酶DEPC水中,使其浓度为5nmol/ml,将等体积的壳寡糖溶液与其混合,壳寡糖的浓度调节为与siRNA溶液的质量比为90,快速混合后涡旋45秒,室温下静置30min通过DLS、TEM观察纳米粒的粒径和形态。
苯硼酸枝接的壳寡糖纳米粒的物理性质:通过动态光散射法测定所知,纳米粒粒径为98.45nm、PDI为0.148,表面带26.2mV的正电荷(图5);电镜下观察纳米粒为球形(图6)。说明利用本专利可以制备载siRNA且物理性质良好的球形纳米粒。
实施例3:PBA-COS/siRNA纳米粒在不同pH下的粒径分布图
将制备的纳米粒与不同pH的HEPES缓冲液混合,通过DLS观察纳米粒粒径的变化。粒径变化总结为表2。从图7可以观察出,随着pH降低,纳米粒的粒径逐渐变大,PDI也逐渐变大,这说明在酸性环境下,siRNA与PBA之间的共价键逐渐断裂,使得纳米粒结构变得松散导致粒径增大、PDI变大。说明本专利能够通过静电作用和化学结合双重作用来实现对带负电siRNA药物的包载和压缩,并且在酸性条件下化学反应逆向进行,能够快速释放siRNA药物。
表2不同pH条件下纳米粒的粒径及PDI变化
pH | 粒径(nm) | 分散系数 |
7.4 | 116.10 | 0.09 |
6.5 | 128.30 | 0.19 |
5.0 | 147.13 | 0.30 |
实施例4:PBA-COS/siRNA纳米粒体外细胞摄取
以Cy5为荧光探针,连接到siRNA上。将适量对数生长期的B16F10细胞接种于24孔板中,继续培养24h后,介质更换为不含血清的培养液进行转染,将提前制备好的纳米粒溶液加入每孔,使每孔siRNA浓度为100nM。设置阴性对照组、裸siRNA组、未枝接的壳寡糖纳米粒组和苯硼酸枝接的壳寡糖纳米粒组,阳离子脂质体2000脂质复合物组作为阳性对照组,每组设置3个复孔。转染4h后,将细胞用PBS冲洗三遍,胰酶消化收集细胞并重悬至0.5mlPBS中,通过流式细胞仪检测细胞对纳米粒摄取效率。
从图8可以看出,利用本专利制备的纳米粒细胞摄取效率高,苯硼酸的引入增加了纳米粒的细胞摄取能力,说明本专利是一种递送效率高的载体。
实验例5:PBA-COS/siRNA纳米粒体外基因沉默
将适量对数生长期的4T1luc细胞接种于24孔板中,继续培养24h后,介质更换为不含血清的培养液进行转染,选取取代度为19.1%的苯硼酸枝接的壳寡糖衍生物制备纳米粒,将制备好的纳米粒溶液加入每孔,使每孔siRNA浓度为100nM。设置阴性对照组、裸siRNA组、未枝接的壳寡糖纳米粒组和苯硼酸枝接的壳寡糖纳米粒组,阳离子脂质体2000脂质复合物组作为阳性对照组,每组设置3个复孔,表3总结了不同组别的荧光素酶活性。
表3 PBA-COS/siRNA纳米粒体外沉默内源性基因情况
组别 | 荧光素酶基因活性 |
空白 | 99.78±8.74 |
裸siRNA | 98.73±4.66 |
COS/siRNA | 78.77±4.30 |
PBA-COS/siRNA | 60.92±4.01 |
Lip2000/siRNA | 54.77±5.01 |
表3及图9表明,利用苯硼酸枝接的壳寡糖衍生物对siRNA进行包载,能够显著提高基因递送效率,达到更好的基因沉默效果,并且与脂质体2000效果无明显差异,说明本专利在基因递送方面具有优势。
实验例6:细胞增殖抑制
将苯硼酸枝接的壳寡糖衍生物冻干粉用培养液稀释成预定浓度的溶液。每个浓度设6个复孔,并设阴性对照组和空白对照组。将适量对数生长期的细胞接种于96孔板中,继续培养48h后,介质以含有20、50、100、200、500μg/ml浓度材料的新鲜培养液替换,分别继续培养48h。每孔加入CCK-8试剂20μl。继续孵育2h,于450nm处测定吸收值,并以650nm为参考波长。表4为苯硼酸枝接的壳寡糖衍生物对小鼠黑色素瘤B16F10细胞的增殖抑制情况。同时利用此方法评价纳米粒体外抑制黑色素瘤增殖情况。计算细胞活力,公式如下:
细胞活力(%)=[(OD实验-OD空白对照)/(OD阴性对照-OD空白对照)]×100
表4苯硼酸枝接的壳寡糖衍生物对B16F10细胞的增殖抑制
图10-a及表4表明,苯硼酸枝接的壳寡糖衍生物在所测定的浓度范围内,B16F10细胞存活率均在90%以上,证实苯硼酸的引入并没有显示出细胞毒性。说明本专利提供的苯硼酸枝接的壳寡糖衍生物在安全性方面存在优势;同时图10-b表明,利用本专利制备的纳米粒体外抑制了黑色素瘤细胞的增值。实验例7:PBA-COS/siRNA纳米粒体内抗黑色素瘤生 长及转移
取4~6周龄的C57BL/6小鼠,建立小鼠体内黑色素瘤模型,待肿瘤体积长到70mm3,将小鼠随机分为5组,每组5只,选取取代度为19.1%的苯硼酸枝接的壳寡糖衍生物制备纳米粒,5组分别瘤内注射100μl生理盐水、裸siRNA、PBA-COS/siNC、COS/siSur和PBA-COS/siSur纳米粒溶液。
以0.3mg siRNA/kg小鼠体重的剂量给药,每两天一次,共计给药四次。每两天检测小鼠的体重和肿瘤体积变化,直到最后一次给药后2天,脱颈处死全部小鼠,解剖肿瘤组织并称重;解剖肺、肝组织于中性甲醇中固定并H&E方法观察肿瘤转移情况。
从图11可以看出,裸siRNA组、阴性对照组的小鼠肿瘤生长情况与生理盐水无明显差别,说明材料本身无治疗效果,并且直接递送siRNA药物无治疗效果;PBA-COS/siRNA纳米粒相比生理盐水组,能够显著抑制小鼠黑色素瘤的肿瘤生长,并且效果优于无苯硼酸枝接的壳寡糖递送siRNA组,这表明苯硼酸的引入进一步增强了抗肿瘤生长作用。
从图12可以看出,在抗转移结果中,PBA-COS/siRNA纳米粒治疗的小鼠肺泡结构完整,无充血现象,且没有发现肿瘤结节,证明利用苯硼酸枝接的壳寡糖递送基因治疗小鼠黑色素瘤可以抑制肿瘤的肺转移。以上说明利用本专利构建基因药物递送系统在体内有良好的抗肿瘤生长及转移的优势。
Claims (8)
1.一种苯硼酸枝接的壳寡糖衍生物或其药学上可接受的盐,其特征在于,该衍生物由苯硼酸类化合物与壳寡糖分子链上的伯胺基通过化学键连接而形成,
所述的苯硼酸枝接的壳寡糖衍生物制备方法包括以下步骤:1)将苯硼酸类化合物、壳寡糖溶于有机试剂,70℃下搅拌回流反应24h;2)将有机试剂蒸发除去后,选用一定截留分子量的透析袋进行透析:在去离子水中透析纯化产物;3)液体冷冻干燥后得冻干粉备用,得到含有苯硼酸枝接的壳寡糖衍生物;所述壳寡糖与苯硼酸类化合物的摩尔比为1:3.4~1:13.6,
所选用的苯硼酸类化合物为2-溴甲基苯硼酸、3-溴甲基苯硼酸或4-溴甲基苯硼酸。
2.根据权利要求1的苯硼酸枝接的壳寡糖衍生物或其药学上可接受的盐,其特征在于,将与苯硼酸类化合物连接的仲胺基的摩尔数占壳寡糖中原有伯胺基总摩尔数的程度定义为苯硼酸的取代度,苯硼酸的取代度为5%~80%。
3.根据权利要求1的苯硼酸枝接的壳寡糖衍生物或其药学上可接受的盐,其特征在于,所选用的壳寡糖分子量为800~10000Da。
4.根据权利要求1所述的苯硼酸枝接的壳寡糖衍生物或其药学上可接受的盐,其特征在于,所述的有机试剂选自无水甲醇、乙醇、二甲基甲酰胺或二甲基亚砜。
5.权利要求1所述的苯硼酸枝接的壳寡糖衍生物或其药学上可接受的盐作为载体在制备体外和体内递送药物中的应用。
6.据权利要求5所述的苯硼酸枝接的壳寡糖衍生物或其药学上可接受的盐的应用,其特征在于,该衍生物作为其中一个组分,配合使用其他非主药成分,制备含有该衍生物的纳米制剂,纳米制剂加载活性药物。
7.根据权利要求6所述的苯硼酸枝接的壳寡糖衍生物或其药学上可接受的盐的应用,其特征在于,所述的纳米制剂中加载的药物包括带负电的基因药物。
8.根据权利要求7所述的苯硼酸枝接的壳寡糖衍生物或其药学上可接受的盐应用,其特征在于,所述的带负电的基因药物选自siRNA、双链RNA或DNA片段。
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