CN116942842A - 一种键合促胞葬功能核酸的纳米银及其制备方法与应用 - Google Patents
一种键合促胞葬功能核酸的纳米银及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种键合促胞葬功能核酸的纳米银及其制备方法与应用,该纳米银为表面修饰促胞葬核酸CpGODN1826的纳米银。使用柠檬酸盐法还原硝酸银得到纳米银,再在含柠檬酸盐的低pH的条件下,将CpGODN1826通过Ag‑S键键合到AgNPs的表面得到CpG‑AgNPs。本发明所述CpG‑AgNPs的制备过程简单,可重复性好,制备得到的CpG‑AgNPs可以在体外发挥多种作用,包括促进巨噬细胞胞葬作用、清除ROS、抗炎和促巨噬细胞重极化。所述CpG‑AgNPs经静脉注射用于动脉粥样硬化治疗时,其组分CpGODN1826和AgNPs可以分别通过促巨噬细胞胞葬和促巨噬细胞M1‑M2重极化协同发挥抗动脉粥样硬化作用,为动脉粥样硬化的治疗提供了一种新的治疗手段。
Description
技术领域
本发明涉及纳米材料和纳米生物医药领域,具体涉及一种键合促胞葬功能核酸的纳米银及其制备方法与应用。
背景技术
动脉粥样硬化是免疫系统参与的炎症性血管性疾病,由动脉血管内皮损伤、脂蛋白渗入并沉积于动脉内膜下、及触发动脉壁的慢性炎症所致,是诱发多种心血管疾病的共同病理基础。斑块形成是动脉粥样硬化的重要病理特征,也是心血管疾病的独立危险因素。晚期动脉粥样硬化斑块可侵入动脉管腔,阻碍血液流动;而斑块脱落可形成血栓,造成更为严重的组织缺血。目前针对动脉粥样硬化的药物治疗以降脂治疗为主,如他汀类药物、PSK9抑制剂等。然而,即使患者达到了降脂的目标治疗水平,仍有心血管事件发生的风险。这是由于降脂治疗虽然可以延缓斑块进展,但是对于已形成的斑块作用较小,因此需要开发新的药物用于已有斑块的清除治疗。
动脉粥样硬化斑块的清除主要涉及巨噬细胞胞葬作用。胞葬作用是机体内一种由“eat me”信号介导的吞噬细胞(主要为巨噬细胞)清除凋亡细胞的高度保守的生理过程。胞葬作用通过清除凋亡细胞,可避免死亡细胞聚集和坏死成分释放,维持机体内稳态。动脉粥样硬化斑块主要由凋亡细胞、泡沫细胞及脂质堆积而成,其中凋亡细胞高表达“don't eatme”信号CD47,导致巨噬细胞胞葬作用受损。由此,动脉粥样硬化病变部位的凋亡细胞不能被及时清除,导致了斑块形成和进一步的扩展。因此,发现一种新的药物,使AS受损的胞葬作用得以恢复,用于动脉粥样硬化的治疗具有重要意义。
发明内容
为解决上述技术问题,本发明提出了一种键合促胞葬功能核酸纳米银的制备方法及应用,即使用柠檬酸盐法还原硝酸银得到纳米银,再在含柠檬酸盐的低pH的条件下,将CpG ODN 1826通过Ag-S键键合到AgNPs的表面得到CpG-AgNPs。经AgNPs递送的CpG ODN1826可以增强巨噬细胞对高表达CD47凋亡细胞及正常凋亡细胞的胞葬作用,同时增加对泡沫细胞的吞噬,有利于清除动脉粥样硬化斑块。同时,载体AgNPs本身还可发挥抗炎、清除ROS和促进巨噬细胞由促炎M1向抗炎M2转化的作用,有利于缓解动脉粥样硬化斑块的病理状态。由此,CpG-AgNPs中的组分CpG ODN 1826和AgNPs可通过发挥各自的药理学活性以协同治疗动脉粥样硬化。
为达到上述目的,本发明首先提供一种键合促胞葬功能核酸的纳米银,包括功能核酸CpG ODN 1826和纳米银AgNPs,所述CpG ODN 1826通过Ag-S键键合到AgNPs的表面修饰得到键合促胞葬功能核酸的纳米银CpG-AgNPs。
作为优选,所述CpG ODN 1826序列如SEQ ID NO.1所示。
作为优选,所述CpG ODN 1826与AgNPs的摩尔比为200:1。
基于一个总的发明构思,本发明还提供了一种键合促胞葬功能核酸的纳米银的制备方法,包括以下步骤:
S1、制备纳米银:将柠檬酸盐、NaBH4、AgNO3与水混合在冰水浴中反应,再加入NaBH4,继续冰水浴反应后得到AgNPs。
S2、制备CpG ODN 1826修饰的AgNPs:在S1步骤中得到的AgNPs加入HS-CpG,涡旋混匀;再加入柠檬酸盐溶液溶液,加入后立马涡旋,瞬时离心,室温静置避光孵育,再次往体系中加入柠檬酸盐溶液,加入后立马涡旋,瞬时离心,室温静置避光孵育,孵育结束后得到CpG-AgNPs。
作为优选,所述S1步骤中柠檬酸盐、NaBH4、AgNO3的摩尔比为3:11:1。
作为优选,所述S2步骤中柠檬酸盐溶液pH为3。
作为优选,所述S2步骤中柠檬酸盐溶液为柠檬酸钠。
作为优选,所述S2步骤第一次室温静置避光孵育时间为5min,第二次室温静置避光孵育时间为30min。
基于一个总的发明构思,本发明还提供了一种键合促胞葬功能核酸的纳米银在制备治疗动脉粥样硬化药物中的应用。
本发明CpG-AgNPs治疗动脉粥样硬化的机理为:
将CpG ODN 1826通过Ag-S键键合到AgNPs的表面得到CpG-AgNPs,经AgNPs递送的CpG ODN 1826可以增强巨噬细胞对高表达CD47凋亡细胞及正常凋亡细胞的胞葬作用,同时增加对泡沫细胞的吞噬,有利于清除动脉粥样硬化斑块。同时,载体AgNPs本身还可发挥抗炎、清除ROS和促进巨噬细胞由促炎M1向抗炎M2转化的作用,有利于缓解动脉粥样硬化斑块的病理状态。由此,CpG-AgNPs中的组分CpG ODN 1826和AgNPs可通过协同发挥其药理学活性对动脉粥样硬化具有较好的疗效。
与现有技术相比,本发明具有以下有益效果:
1、本发明提供的键合促胞葬功能核酸纳米银CpG-AgNPs,其组分CpG ODN 1826可以增强巨噬细胞对高表达CD47凋亡细胞及正常凋亡细胞的胞葬作用,同时增加对泡沫细胞的吞噬,有利于清除动脉粥样硬化斑块;与此同时,其组分载体AgNPs本身可发挥抗炎、清除ROS和促进巨噬细胞由促炎M1向抗炎M2转化的作用,在体内降低炎症因子TNFα和IL6的水平,有利于缓解动脉粥样硬化斑块的病理状态。因此,CpG-AgNPs可以通过CpG ODN 1826和AgNPs协同以发挥治疗动脉粥样硬化的作用。
2、CpG-AgNPs中的AgNPs对CpG ODN 1826具有良好的保护作用,可减少CpG ODN1826被体内酶降解,保持CpG ODN 1826在体循环中的稳定,进入细胞后才会释放以发挥作,且在细胞中释放后可在溶酶体中可长时间停留,有利于其与受体结合以更好的发挥生物学效应;同时,AgNPs在一定程度上能降低血浆中的TG和LDL,CpG-AgNPs可以减少斑块厚度和坏死核心面积,同时可以通过减少巨噬细胞浸润、降低MMP9的表达和增加α-SMA表达和胶原含量,提高斑块稳定性。
3、本发明通过使用柠檬酸盐法还原硝酸银得到纳米银,再在含柠檬酸盐的低PH的条件下,将CpG ODN 1826通过Ag-S键键合到AgNPs的表面得到CpG-AgNPs,制备过程简单,简单可控。
4、本发明提供的CpG-AgNPs分散性良好,胶体稳定性良好,CpG ODN 1826在AgNPs上的吸附可在30min内稳定;能在生物环境中保持稳定,具有良好的生物安全性,有利于其应用发展。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实验例1中CpG-AgNPs的表征结果;图1A为CpG-AgNPs的制备流程图;图1B为CpG ODN 1826吸附到AgNPs上的吸附动力学结果;图1C为CpG ODN 1826吸附到每个AgNP上不同投料比的吸附结果;图1D为AgNPs和CpG-AgNPs的电位结果;图1E为AgNPs和CpG-AgNPs的紫外光谱图;图1F为AgNPs和CpG-AgNPs的透射电镜图;图1G~H为AgNPs和CpG-AgNPs在不同介质中的粒径变化结果;图1I为AgNPs载体对CpG的保护作用结果;图1J为CpG-AgNPs在含GSH和不含GSH条件下CpG ODN 1826的释放结果;
图2为本发明对比例1的CpG-AgNPs和NC-AgNPs纳米粒对不同细胞存活率影响的结果,包括RAW264.7、MOVAS、HUVEC细胞;图2A为CpG-AgNPs的细胞毒性结果;图2B为NC-AgNPs的细胞毒性结果;
图3为本发明实验例2检测获得的游离CpG ODN 1826和CpG-AgNPs纳米粒的细胞摄取结果,图3A为显微镜拍照结果;图3B为流式检测的结果;图3C是图3B的定量结果;
图4为本发明实验例3检测检测获得CpG-AgNPs与溶酶体共定位的激光共聚焦图;
图5为本发明实验例4检测获得CpG-AgNPs促进胞葬作用的结果图,图5A吞噬的靶细胞为高表达CD47的凋亡MOVAS细胞;图5B吞噬的靶细胞为正常凋亡MOVAS细胞;图5C吞噬的靶细胞为泡沫MOVAS细胞;图5D-F分别为图5A-C的定量结果;
图6为本发明实验例5检测获得的CpG-AgNPs清除ROS,抗炎及促M1-M2复极化作用结果,图6A为CpG-AgNPs清除ROS的胞内显微拍照的结果;图6B为流式检测胞内ROS清除的结果,图6C为图6B的定量分析;图6D-F为qPCR检测CpG-AgNPs抗炎作用结果,图6D为IL-1β,图6E为TNFα,图6F为IL6;图6G为流式检测巨噬细胞极化相关分子CD80和CD206的代表性结果,图6H为CD80表达的定量分析;图6I为CD206表达的定量分析;
图7为本发明实验例6中检测获得CpG-AgNPs体内抗AS结果,图7A为动物实验流程图;图7B、C为给药期间动物体重变化情况;图7D-G分别为AS小鼠血脂四项检测结果,包括TG(D)、CHO(E)、HDL(F)、LDL(G);
图8为本发明实验例6中小鼠主动脉窦切片检测,图8A是主动脉窦切片代表性的油红O染色、HE染色、Mac-3免疫组化、MMP9免疫组化、Masson染色、α-SMA免疫组化的结果;图8B-G分别为以上结果的定量分析;
图9为本发明实验例6中检测获得CpG-AgNPs体内抗AS治疗机制的结果,图9A主动脉窦切片Mac-3和caspase-3免疫荧光双染结果;图9B为图9A的定量结果;图9C为主动脉窦切片CD80和CD206的免疫荧光双染结果;图9D和图9E分别为体内TNF-α和IL-6炎症因子水平检测结果;
图10为本发明实验例7检测获得的CpG-AgNPs体内安全性结果,图10A-D分别为生化指标CRE、BUN、AST、ALT检测结果;图10E为各组小鼠主要脏器(心、肝、脾、肺、肾)的HE染色结果;
图11为本发明CpG-AgNPs的作用机理图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段;若未特别指明,实施例中所用试剂均为市售。
实施例1
制备纳米银(AgNPs)
提前准备好洁净圆口烧瓶,依次往其中加入390.8mL水,1.2mL100 mM柠檬酸钠,4mL100mM NaBH4,4mL 10mMAgNO3,冰水浴反应30min后,加入0.4mL100 mM NaBH4,继续冰水浴反应10min,得到AgNPs,4℃避光保存以待后续使用。
实施例2
制备CpG ODN 1826修饰的AgNPs(CpG-AgNPs)
50μL 20nMAgNPs体系,往其中加入2μL 100μM HS-CpG,涡旋混匀;之后加入0.5μL500mM柠檬酸三钠(pH=3),加入后立马涡旋,瞬时离心,室温静置避光孵育5min,再次往体系中加入0.5μL 500mM柠檬酸三钠(pH=3),加入后立马涡旋,瞬时离心,室温静置避光孵育30min,孵育结束后得到CpG-AgNPs,合成原理如图1A所示。
实施例3
制备CpG ODN 1826阴性对照序列修饰的AgNPs(NC-AgNPs)
50μL 20nMAgNPs体系,往其中加入2μL 100μM HS-NC(其中CpG ODN 1826发挥功能的CG碱基由GC碱基替代,序列为TCCATGAGCTTCCTGAGCTT),涡旋混匀;之后加入0.5μL 500mM柠檬酸三钠(pH=3),加入后立马涡旋,瞬时离心,室温静置避光孵育5min,再次往体系中加入0.5μL 500mM柠檬酸三钠(pH=3),加入后立马涡旋,瞬时离心,室温静置避光孵育30min,孵育结束后得到NC-AgNPs。
实验例1
一、考察CpG ODN 1826吸附时间
在实施例2条件下,加入HS-CpG-FAM,分别在不同时间点取上清,检测荧光以计算CpG ODN 1826在AgNPs上的吸附,由此得到CpG ODN 1826在AgNPs上的吸附动力学
结果如图1B所示,随着时间的延长,荧光强度逐渐降低并稳定下来,CpG ODN 1826在AgNPs上的吸附可在30min内稳定。
二、考察CpG ODN 1826与AgNPs投料比例
在实施例2条件下,加入不同量的HS-CpG-FAM,分别在孵育结束后取上清测荧光,以计算CpG ODN 1826在AgNPs上的吸附量。
结果如图1C所示,HS-CpG与AgNPs的投料比为200:1时,CpG ODN 1826在AgNPs上的吸附量趋于稳定,每个AgNP上约可吸附64个CpG ODN 1826。因此选择制备CpG-AgNPs时,HS-CpG与AgNPs的投料比为200:1。
三、电位检测
在实施例1-2条件下,取制备得到的AgNPs和CpG-AgNPs溶液;取两种溶液分别置于Marlven Nano ZS仪器,检测电位,测定池温度设定为25℃
结果如图1D所示,AgNPs电位约为-7mV;CpG-AgNPs电位约为-14mV。由于CpG ODN1826带负电,因此吸附CpG ODN 1826后AgNPs负电位的绝对值更大,说明CpG ODN 1826成功吸附到AgNPs表面。
四、紫外吸收光谱扫描
分别对AgNPs和CpG-AgNPs进行紫外光谱扫描,以空白溶剂为对照,扫描300-600nm的紫外吸收光谱。
测定结果如图1E所示,AgNPs和CpG-AgNPs纳米粒均在400nm左右有特征吸收峰,说明吸附CpG ODN 1826的AgNPs和裸AgNPs无明显性质差异。
五、形态观察
观察AgNPs和CpG-AgNPs的形态,观察形态的检测方法:样品滴加在覆盖碳膜的铜网上,置于干燥箱中,置于干燥器中干燥,重复2~3次,然后将其置于透射电镜Titan G260-300下,观察其形态。
结果如图1F所示,从图1F中可知:本发明的CpG-AgNPs与裸Ag NPs相比分布无明显差异,单一粒子呈类球形,粒径较小。
六、胶体稳定性考察
将实施例1~2制备的AgNPs和CpG-AgNPs分别置于FBS、10%1640、0.9%NaCl及PBS这四种不同介质中,分别在0、2、4、8、12、24h测定他们的粒径,以评估其稳定性。
结果如图1G和图1H所示:CpG-AgNPs在各种介质中均稳定,粒径无较大变化,而裸AgNPs在0.9%NaCl和PBS中粒径明显增大,说明AgNPs在这两种介质中不稳定。可见相较于裸AgNPs,CpG-AgNPs具有更好的胶体稳定性,可在生物环境中保持稳定。
七、AgNPs对CpG ODN 1826的保护作用
测定方法如下:按照实施例2的方法,使用HS-CpG-FAM制备得到的CpG-AgNPs,另外配制同等终浓度的游离FAM-CpG,分别与终浓度为0U/mL、10U/mL、100U/mL的DNase I孵育,之后再往体系加入EDTA,65℃水浴使DNase I失活。取上清与DNA loading buffer混合,以游离的FAM-CpG为对照,上样跑尿素胶,之后荧光显影,观察降解程度。
结果如图1I所示,CpG-AgNPs组仅在最高100U/mL的DNase I浓度下有少量降解,而游离CpG组在DNase I为100U/mL的浓度下全部降解完全,说明AgNPs对CpG ODN 1826具有良好的保护作用,可减少CpG ODN 1826被体内酶降解。
八、CpG-AgNPs在不同介质中的释放
按照实施例2的方法,使用HS-CpG-FAM制备得到的CpG-AgNPs,离心浓缩后,分别加入PBS(PH7.4)、PBS+GSH(PH7.4)缓冲液重悬,之后在37℃,100rpm摇床下孵育,分别在0、0.5、1、2、4、8、12、24h取部分体系溶液,取上清测荧光。
结果如图1J所示,CpG-AgNPs在体循环条件下保持稳定,仅有10%CpG ODN 1826释放;而在胞内含有GSH的条件下,24内释放可达80%;说明其体循环中稳定,进入细胞后才会释放以发挥作用。
对比例1
考察CpG-AgNPs及NC-AgNPs的细胞毒性
根据实施例2的条件制备CpG-AgNPs纳米粒,根据实施例3的条件制备NC-AgNPs纳米粒。分别将三种细胞(RAW264.7、MOVAS、HUVEC)分别提前接种在96孔板中,再分别用0、0.1、0.2、0.4、0.8、1.6nM的CpG-AgNPs、NC-AgNPs处理各细胞48h,然后去除CpG-AgNPs、NC-AgNPs孵育液;再将各处理后的细胞分别与MTT工作液在37℃条件孵育4h,再将MTT工作液去除;每孔加入100μL DMSO以溶解生成的结晶。最后用酶标仪测量490nm处吸光度来以评价CpG-AgNPs、NC-AgNPs的细胞毒性。
检测结果如图2所示,CpG-AgNPs(A)、NC-AgNPs(B)即使在最高的给药浓度1.6nM的条件下,三种细胞(RAW264.7、MOVAS、HUVEC)的细胞存活率都大于80%,说明CpG-AgNPs及NC-AgNPs纳米粒具有良好的生物安全性。
实验例2
考察CpG-AgNPs的细胞摄取情况
使用HS-CpG-FAM,根据实施例2的条件制备FAM-CpG-AgNPs。提前将RAW264.7细胞种板到24孔板中,使用游离FAM-CpG,FAM-CpG-AgNPs分别处理RAW264.7细胞4h,以正常培基组的细胞为对照,孵育结束后分别用显微镜拍照和流式细胞术检测各细胞对CpG的摄取结果。(1)通过显微镜观察细胞摄取:游离FAM-CpG及FAM-CpG-AgNPs孵育结束后,弃掉原培基,用PBS洗涤细胞3次后;再用多聚甲醛与细胞孵育15min以固定细胞;固定结束后,用PBS洗3次细胞,再加入hoechst 33342染色液与细胞孵育5min以给细胞核染色,hoechst 33342染色液加入的量以能完全覆盖为宜;染色结束后,PBS洗3次;最后往细胞中加入PBS,在显微镜观察。(2)使用流式细胞术检测细胞摄取:游离FAM-CpG及FAM-CpG-AgNPs孵育结束后,弃掉原培基,PBS洗涤细胞3次后,各孔加入同等量的PBS,之后用枪头将将处理后的RAW264.7细胞吹下收集到2mL EP管中,上机用流式细胞仪进行检测。
显微镜观察结果如图3A所示,流式细胞仪检测结果如图3B所示,图3C是对图3B的定量分析。由此可知,与游离FAM-CpG相比,FAM-CpG-AgNPs处理组中的细胞荧光更强,说明载体AgNPs可有效递送CpG ODN 1826至巨噬细胞RAW264.7中。
实验例3
检测CpG-AgNPs与溶酶体定位的情况
按照实施例2的方法,使用HS-CpG-FAM制备得到的FAM-CpG-AgNPs。提前一晚将RAW264.7细胞接种至激光共聚焦小皿里,分别用FAM-CpG-AgNPs孵育细胞,时间为2h、4h、12h。之后,用PBS洗涤3次细胞。再往各激光共聚焦小皿中加入溶酶体探针,避光孵育30min以染色溶酶体;去除溶酶体探针后,再用PBS洗3次细胞,用多聚甲醛孵育细胞15min以固定细胞(避光,室温);再用PBS洗3次细胞,往每个小皿中分别加入DAPI染色工作液,加入的量以能完全覆盖细胞为宜,孵育5min(避光,室温);PBS洗3次细胞后,每孔加入一定量的PBS,将小皿放于激光共聚焦显微镜下观察。
观察结果如图4所示,从图中可以看出,溶酶体的荧光和CpG的荧光几乎全部重合,说明CpG-AgNPs上的CpG ODN 1826,在细胞中释放后可在溶酶体中可长时间停留。由于CpG受体TLR9在溶酶体中,CpG ODN 1826在溶酶体的停留有利于其与受体结合以更好的发挥生物学效应。
实验例4
考察CpG-AgNPs的促胞葬作用
提前将MOVAS细胞种过夜,先用TNF-α处理诱导CD47高表达,之后加入十字孢碱诱导高表达CD47的细胞凋亡,得到CD47高表达的凋亡MOVAS细胞。仅用十字孢碱诱导细胞凋亡,获得正常凋亡的MOVAS细胞。此外,用oXLDL与MOVAS细胞共孵育,得到泡沫MOVAS细胞。因此,得到被巨噬细胞吞噬的三种靶细胞。将CD47高表达的凋亡MOVAS细胞、正常凋亡MOVAS细胞、泡沫MOVAS细胞分别用cell tracker deep red染料标记为深红色荧光,分别作为被吞噬的靶细胞。同时,提前种板RAW264.7巨噬细胞,分别用CpG-AgNPs和NC-AgNPs处理细胞,以NC-AgNPs处理(用CpG的阴性对照序NC序列制备得到的NC-AgNPs)为对照。制剂处理结束后,用cell tracker CMFDA染料将巨噬细胞RAW264.7标记为绿色荧光作为吞噬细胞。最后,将标记绿色荧光的吞噬细胞分别与标记深红色荧光的被三种被吞噬靶细胞分别在无血清条件下共孵育2h后,分别收集细胞进行流式细胞术检测,其中双阳性细胞所占比例就是RAW264.7细胞对靶细胞的吞噬率。
结果如图5所示,图5A、图5B、图5C分别为RAW264.7细胞对高表达CD47的凋亡MOVAS细胞、正常凋亡MOVAS细胞及泡沫MOVAS细胞的吞噬检测结果;图5D、图5E、图5F分别为图5A、5B、图5C的定量分析。由此可见,相比与NC-AgNPs,CpG-AgNPs纳米粒可以促进巨噬细胞的胞葬作用,增加对凋亡细胞及泡沫细胞的吞噬,说明键合在AgNPs的CpG ODN 1826具有促胞葬的作用。
实验例5
考察CpG-AgNPs胞内清除ROS、抗炎以及促M1-M2巨噬细胞重极化效应
考察CpG-AgNPs胞内清除ROS的实验步骤如下:按照实施例2的条件制备得到CpG-AgNPs。提前将RAW264.7细胞种板在6孔板中,之后使用脂多糖(LPS,10μg/mL)诱导48h,使RAW264.7胞内ROS过载。而后,移除LPS,各孔分别用LPS、转染游离NC序列、转染游离CpG序列、NC-AgNPs及CpG-AgNPs分别处理。处理结束后各细胞与DCFH-DA荧光探针共孵育30min,之后去除ROS探针,PBS洗三遍。处理结束后,细胞直接用荧光显微镜观察制剂的ROS清除效果;并收集细胞进行流式细胞术检测以定量分析制剂对ROS清除效果。考察CpG-AgNPs的抗炎和促M1-M2巨噬细胞重极化作用,其实验步骤如下:RAW264.7细胞先提前种板到孔板中使其贴壁,之后用LPS(10μg/mL)刺激48h,从而使巨噬细胞向M1型极化。之后分别用LPS、转染游离NC序列、转染游离CpG序列、NC-AgNPs及CpG-AgNPs分别处理RAW264.7细胞。处理结束后,收集细胞提取RNA进行qPCR检测,以评估制剂对炎症因子的影响。另外收集各处理后的细胞,分别与M1巨噬细胞典型分子CD80和M2巨噬细胞典型分子CD206分别共孵育,收集细胞进行流式细胞术检测CD80和CD206的表达情况,以评估制剂促巨噬细胞由M1向M2转化的效果。
结果如图6所示,图6A为使用DCFH-DA探针检测胞内ROS清除的显微拍照结果;图6B、C为流式细胞术检测胞内ROS清除的结果,图6C为图6B的定量分析。图6D-F为qPCR检测各处理后的细胞炎症因子表达水平结果,IL-1β(D),TNF-α(E),IL-6(F)。图6G流式细胞术检测巨噬细胞极化相关分子CD80和CD206的结果,图6H为CD80表达的定量分析;图6I为CD206表达的定量分析。由此可见,CpG-AgNPs可清除胞内ROS;降低炎症因子IL-1β、TNF-α、IL-6的表达;同时,CpG-AgNPs处理细胞后后,M1型的代表性分子CD80表达下降,M2型的代表性分子CD206表达上升,说明CpG-AgNPs可促使巨噬细胞由促炎的M1表型转变为抗炎M2型。
实验例6
考察CpG-AgNPs体内抗动脉粥样硬化的效果
ApoE-/-小鼠(6周龄)用高脂饮食喂养4周以诱导动脉粥样硬化发生,之后随机将ApoE-/-小鼠分为4组,分别为生理盐水组、游离CpG、NC-AgNPs和CpG-AgNPs组。通过尾静脉注射对小鼠进行给药,每5天注射一次,共注射15次,同时伴随高脂饮食。给药开始后,小鼠体重每周记录。给药结束后,对小鼠实施安乐死后采集血液、腹主动脉和主要脏器。测定了各处理组小鼠的血脂四项,包括胆固醇(CHO)、甘油三酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)。同时用ELISA试剂盒检测了血浆中炎性细胞因子,包括IL6、TNFα。此外,通过用油红O和HE染色主动脉窦的冰冻切片来评估斑块厚度和坏死核心。同时为了评估斑块稳定性,进行了免疫组化染色(包括Mac-3、MMP-9、α-SMA)和masson染色。此外,为考察体内的胞葬情况,进行了Mac-3和caspase-3免疫荧光双染,游离凋亡细胞用星号表示,巨噬细胞相关的凋亡细胞用箭头表示。为考察体内巨噬细胞的重极化的情况,进行CD80和CD206的免疫荧光双染。
结果如图7-9所示,图7为本发明实验例7检测获得CpG-AgNPs体内抗AS结果。图7A为动物实验流程图;图7B、C为给药期间动物体重变化情况,说明各组处理均可以在一定程度上降低小鼠体重,其中CpG-AgNPs效果最为明显;图7D-G分别为AS小鼠血脂四项检测结果,包括TG、CHO、HDL、LDL,说明载体AgNPs在一定程度上能降低血浆中的TG和LDL。
图8为小鼠主动脉窦切片检测,图8A为主动脉窦切片代表性的油红O染色、HE染色、Mac-3免疫组化、MMP9免疫组化、Masson染色、αSMA免疫组化的结果,图8B-G分别为以上结果的定量分析;由此可见,CpG-AgNPs可以减少斑块厚度和坏死核心面积,同时可以通过减少巨噬细胞浸润、降低MMP9的表达和增加α-SMA表达和胶原含量,提高斑块稳定性。图9为CpG-AgNPs体内抗AS治疗机制的结果,图9A主动脉窦切片Mac-3和caspase-3免疫荧光双染结果;图9B为图9A的定量结果;由此可见,经AgNPs递送后的CpG可以在体内明显促进巨噬细胞对凋亡细胞的吞噬。图9C为主动脉窦切片CD80和CD206的免疫荧光双染结果,说明CpG-AgNPs可以在体内下调巨噬细胞CD80的表达,上调巨噬细胞CD206的表达;由此说明CpG-AgNPs可以促进体内巨噬细胞由M1型向M2型转化。图9D和图9E分别为体内TNFα和IL6炎症因子水平检测结果,说明CpG-AgNPs可以在体内降低炎症因子TNFα和IL6的水平。
图11为CpG-AgNPs体内抗动脉粥样硬化的作用机理图。
实验例7
考察CpG-AgNPs体内安全性
按照实验例7的方法处理小鼠后,取小鼠血浆用于肝肾功能指标检测;取小鼠的心、肝、脾、肺、肾,经4%多聚甲醛固定后,通过H&E染色,在光学显微镜下观察病理改变。
结果如图10所示。图10A-D分别为生化指标CRE、BUN、AST、ALT检测结果;图10E为各组小鼠主要脏器(心、肝、脾、肺、肾)的HE染色结果。与模型组相比,各制剂处理组的血生化检测结果均无明显改变,且主要脏器也未见明显病变。说明CpG-AgNPs在体内应用具有良好的安全性。
以上所述实施例,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明的技术范围内,根据本发明的技术方案及其构思加以等同替换或改变,都应涵盖在本发明的保护范围内。
Claims (8)
1.一种键合促胞葬功能核酸的纳米银,其特征在于,包括功能核酸CpGODN1826和纳米银AgNPs,所述CpGODN1826通过Ag-S键键合到AgNPs的表面修饰得到键合促胞葬功能核酸的纳米银CpG-AgNPs。
2.根据权利要求1所述的一种键合促胞葬功能核酸的纳米银,其特征在于,所述CpGODN1826序列如SEQ ID NO.1所示。
3.根据权利要求1所述的一种键合促胞葬功能核酸的纳米银,其特征在于,所述CpGODN1826与AgNPs的摩尔比为200:1。
4.一种如权利要求1-3任一项所述键合促胞葬功能核酸的纳米银的制备方法,其特征在于,包括以下步骤:
S1、制备纳米银:将柠檬酸盐、NaBH4、AgNO3与水混合在冰水浴中反应,再加入NaBH4,继续冰水浴反应后得到AgNPs;
S2、制备CpGODN1826修饰的AgNPs:在S1步骤中得到的AgNPs体系中加入HS-CpG,涡旋混匀;再加入柠檬酸盐溶液,加入后立马涡旋,瞬时离心,室温静置避光孵育,再次往体系中加入的柠檬酸盐溶液,加入后立马涡旋,瞬时离心,室温静置避光孵育,孵育结束后得到CpG-AgNPs。
5.根据权利要求4所述的制备方法,其特征在于,所述S1步骤中柠檬酸盐、NaBH4、AgNO3的摩尔比为3:11:1。
6.根据权利要求4所述的制备方法,其特征在于,所述S2步骤中柠檬酸盐溶液pH为3。
7.根据权利要求4所述的制备方法,其特征在于,所述S2步骤中柠檬酸盐溶液为柠檬酸钠。
8.一种如权利要求1-3任一项所述的键合促胞葬功能核酸的纳米银或如权利要求4~7任一项所述制备方法制得的键合促胞葬功能核酸的纳米银在制备治疗动脉粥样硬化药物中的应用。
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