CN115337397A - 负载siRNA/PEI的PDA纳米药物及其制备方法和用途 - Google Patents
负载siRNA/PEI的PDA纳米药物及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种负载siRNA/PEI的PDA纳米药物的制备方法,包括如下步骤:(1)PEI与siRNA反应10‑120分钟,其中siRNA与PEI的质量比为1:1‑1:10。配制氨水或Tris‑HCl的醇水溶液,醇水溶液中乙醇和水的体积比为1:1‑1:10;在氨水或Tris‑HCl的醇水溶液中加入siRNA/PEI混合物和盐酸多巴胺,siRNA与盐酸多巴胺的质量比为1:1‑1:1000,氨水或Tris‑HCI与盐酸多巴胺的摩尔比为1:1‑1:50,搅拌,用离心法收集PDA纳米药物;(2)靶向分子的修饰:将PDA纳米药物分散于PBS缓冲液中,用EDC/NHS通过氨基和羧基的耦合反应,结合带羧基的靶向分子叶酸,EDC/NHS与盐酸多巴胺的摩尔比为1:1‑1:10,叶酸与盐酸多巴胺的摩尔比为1:1‑1:50,离心收集靶向分子修饰的PDA纳米药物,制得负载siRNA/PEI的PDA纳米药物。此外,本发明还公开了负载siRNA/PEI的PDA纳米药物在制备治疗肝癌的药物中的用途。
Description
技术领域
本发明涉及抗癌基因纳米药物的制备及应用,特别涉及一种负载siRNA/PEI,具有靶向给药及智能响应的聚多巴胺纳米药物,通过敲低促癌基因ROC1,在抗肝癌领域的用途。
背景技术
肝癌是常见的人类致死性恶性肿瘤之一,具有极高的死亡率,严重危害人类健康和生命安全。传统的肝癌药物具有溶解性差、用药剂量大、副作用大等特点,严重制约了在肝癌治疗中的作用效果。因此,人们迫切希望寻找到高效低毒和高选择性的抗癌药物,即能靶向性杀伤肿瘤细胞,对正常细胞的毒性较小,从而发挥最有效的抗肿瘤效果。
Neddylation修饰是一种新发现的蛋白翻译后修饰途径,也是新型抗肿瘤分子靶点蛋白,即将类泛素小分子NEDD8结合到底物分子的过程,其主要功能是调节蛋白活性。Neddylation修饰是一个耗能的级联反应过程,NEDD8在ATP激活的NEDD8活化酶(E1,NAE)催化下,被转移至结合酶(E2,UBE2M或UBE2F),最后在连接酶E3的作用下与CRL蛋白家族连接。CRL是泛素连接酶中最大的蛋白质亚家族,它能通过调控众多蛋白的聚集和合成来调控细胞的生物学功能。迄今研究发现,NEDD8级联反应包括1个E1(由NAE1和UBA3形成的异二聚体),2个E2(UBC12/UBE2M和UBE2F),一些E3s(ROC1/RBX1,RBX2/ROC2/SAG,DCN1-5)(J.Natl.Cancer Inst.2014,106:dju083)。有文献报道,在肝癌病人组织中检测到高度活化的Neddylation,以及高表达的NAE1、UBA3、UBC12及ROC1等,如果敲低NEDD8的活化酶E1(NAE)、结合酶E2及连接酶E3,势必会抑制肝癌组织中Neddylation的活性,从而抑制肝癌的发生发展(Mol.Cell Proteomics.2015,14:499-509)。
RNA干扰是生物中普遍存在的一种自然现象,是由双链RNA启动的序列特异的转录后基因沉默过程,通过导入外源或内源的双链RNA(dsRNA),细胞内加工后可以特异性阻滞基因的表达,使内源性信使RNA(mRNA)发生特异性降解,从而引起转录后基因沉默。具有高效、高特异抑制靶基因表达的作用,但siRNA在体内的易酶解、循环寿命短、细胞内吞和胞质运输受损、组织穿透不足、血脑屏障及非靶向递送,限制在肿瘤治疗方面的应用。目前基因治疗领域主要有三种输送技术,分别是物理转染技术、病毒载药系统和非病毒纳米载体系统。其中物理递送方法比较常用的是电穿孔法,难以在体内使用;病毒载体(慢病毒、腺病毒及腺相关病毒等)是目前使用最广泛的一种方法,但其存在着潜在的安全性和有效性问题,例如对宿主细胞可能产生致癌、致突变的风险,另外病毒载体负载能力有限(比如,最有前景的腺相关病毒只能负载不大于4.7kb的DNA序列)。非病毒纳米载体与病毒载体相比,具有免疫原性低、安全性好等优点。近年来,虽然有络绎不绝的siRNA类药物进入临床试验,但是基于siRNA的不稳定性,绝大部分的药物受阻于Ⅰ-Ⅱ期临床试验。因此,开发高效低毒的抗癌基因纳米药物具有举足轻重的作用。
目前,非病毒纳米载体主要包括阳离子聚合物、脂质体和无机纳米材料等。聚乙烯亚胺(PEI)是一种具有出色转染效果的金标准聚合物,其衍生物也已经运用到多种疾病的临床试验中(GMP invivo-jet PEI)。这是因为PEI可以稳定结合DNA进入细胞,并能通过“质子泵”机制从细胞溶酶体逃逸,释放DNA分子,达到基因转染的目的。但其严重的细胞毒性和不可降解性阻碍了其作为基因传递载体的临床应用。
纳米药物由于其小尺寸效应,可以包埋基因药物(siRNA或Crispr-cas9质粒等),还可以修饰靶向分子及智能化响应基团等,因此具有EPR(实体瘤的高通透性和滞留)效应、延长药物的消除半衰期、靶向递送、可控释放及透过机体屏障(血脑屏障)等优势,在癌症治疗方面取得了长足的发展。近期研究发现,纳米药物通过EPR被动靶向到肿瘤组织的量较少,而且大多数仅停留在肿瘤组织周边。因此,传统的EPR模型过于简单,药物难以富集并渗透到肿瘤组织内部。所以,在纳米药物表面修饰上靶向分子,是纳米药物富集在肿瘤部位及成功透膜的关键所在。常见的靶向分子有小分子(叶酸、凝集素等)、多肽、多糖、抗体及核酸适配体等。叶酸受体(FR)在肿瘤细胞的高表达使得叶酸(FA)成为较为常见的靶向分子。新一代的纳米药物不仅能够选择性地输送抗癌药物到肿瘤组织,而且一旦到达肿瘤组织,还应该能够以可控释放或智能响应的方式释放抗癌药物。研究人员已经探索了许多智能响应型纳米药物,像外部刺激(光、磁场、超声等)和内部刺激(pH、温度、酶、氧化还原电位等)响应。大量研究表明,肿瘤组织pH的水平(6.5-6.9)一般低于同一组织来源的正常对照(7.2-7.4),这主要是因为肿瘤组织无氧代谢(Warburg效应)旺盛,产生大量酸性代谢产物(乳酸等)。基于肿瘤微环境低的pH值,可以设计基于pH智能响应的纳米药物。
光热治疗技术作为一种新型的肿瘤治疗策略,使得具有光热转化能力的有机光敏分子或者无机纳米材料,在近红外光照射下能够转化为热量发挥多种抗肿瘤作用,包括热消融作用、克服化疗耐药作用和抑制肿瘤转移等作用。目前可用于肿瘤光热制剂多为一些有机光敏分子(吲哚菁绿、亚甲基蓝等)或一些无机材料(贵金属纳米粒子、金属硫族化合物纳米材料、碳基纳米材料以及量子点等),但都存在一定的缺点,比如有机光敏分子具有很短的血液半衰期以及不能选择性富集在瘤区,无机材料也存在生物相容性不佳等缺点,这些缺点制约了现有光热制剂的应用。聚多巴胺(PDA)是天然生物色素-黑色素的主要成分,该粒子具有良好的稳定性、生物可降解性、生物相容性和光热转换特性,是一种比较理想的载体材料,但PDA作为基因药物siRNA的载体,其负载能力并不高。
发明内容
本发明所要解决的技术问题是提供一种负载siRNA/PEI的PDA基因纳米药物的制备方法及其在抑制肿瘤中的应用,填补了之前PDA纳米载体没有负载过siRNA/PEI的空缺。
根据实施例,本发明提供的一种负载siRNA/PEI的PDA纳米药物的制备方法,包括如下步骤:
(1)PEI(分子量MW=200-100000)与siRNA反应10-120分钟,制得siRNA/PEI混合物,其中siRNA与PEI的质量比为1:1-1:10;配制氨水或Tris-HCl的醇水溶液,醇水溶液中乙醇和水的体积比为1:1-1:10;在氨水或Tris-HCl的醇水溶液中加入siRNA/PEI混合物和盐酸多巴胺,siRNA与盐酸多巴胺的质量比为1:1-1:1000,氨水或Tris-HCI与盐酸多巴胺的摩尔比为1:1-1:50,搅拌,用离心法收集PDA纳米药物;
(2)靶向分子的修饰:将PDA纳米药物分散于PBS缓冲液中,用EDC/NHS通过氨基和羧基的耦合反应,结合带羧基的靶向分子叶酸,EDC/NHS与盐酸多巴胺的摩尔比为1:1-1:10,叶酸与盐酸多巴胺的摩尔比为1:1-1:50,离心收集靶向分子修饰的PDA纳米药物,制得负载siRNA/PEI的PDA纳米药物。
根据后续实施例和试验例,本发明前述制备方法中之步骤(1)中,耦合反应的温度优选为10-40℃,反应时间优选为1-48小时。
根据后续实施例和试验例,本发明前述制备方法中之步骤(1)、(2)中,离心转速优选为3000rpm-15000rpm,离心时间优选为2-30分钟。
根据实施例,本发明方法制得的负载siRNA/PEI的PDA纳米药物在制备治疗肝癌的药物中的用途,亦即,负载siRNA/PEI的PDA纳米药物应用在肝癌的治疗上。
在本发明中,申请人构建了具有靶向递送和智能响应的PDA纳米药物(图3),该纳米药物,在FA的靶向作用下,胞吞进入肿瘤细胞,溶酶体逃逸后,在肿瘤微环境低的pH值条件下刺激释放基因药物siRNA/PEI,通过抑制ROC1,阻断Neddylation修饰,诱导凋亡因子的集聚;联合PDA的NIR光热治疗,从而达到高效抑制肝癌发生发展的目的。
相对于现有技术,本发明采用较简单的工艺制备合成负载siRNA/PEI的PDA纳米药物,产率高,生物相容性好,具有产业化实施的前景;本发明制备的负载siRNA/PEI的PDA纳米药物,分布均匀,具有良好的胶体稳定性及生物相容性,具有显著的抗肿瘤效果,联合NIR光热治疗,体现增强的治疗效果,具有潜在的应用价值。
附图说明
图1显示Neddylation修饰过程及其主要的抗癌靶点;
图2显示TCGA数据库中(A)ROC1(RBX1)在肝癌组织中高表达;(B)ROC1高表达肿瘤患者的生存(Kaplan-Meier)曲线;
图3为siRNA负载的FA修饰的PDA纳米药物合成及应用示意图;
图4为PDA纳米粒子的(A)TEM图和(B)DLS粒径分布图,标尺100nm;
图5显示PDA纳米粒子对(A)肝正常细胞QSG-7701,(B)肝癌细胞Huh7和(C)肝癌细胞SK-Hep-1的生物相容性,以及NIR光照的生物相容性;
图6显示PDA纳米药物的pH智能响应,以及PDA纳米材料的NIR光热响应现象,(A)Cy3-siRNA负载的PDA纳米药物在pH 5.0以及808激光照射下的可控释放,(B)PBS,PDA纳米载体及siRNA负载的纳米药物在2W·cm-2 808激光器下的NIR光热响应现象;
图7显示Cy3-siRNA负载的聚多巴胺纳米药物的靶向分子FA的特异性递送效果。(A)荧光显微镜分析FA修饰的Cy3-siRNA负载的聚多巴胺纳米药物在肝正常细胞QSG-7701,肝癌细胞Huh7和SK-Hep-1中Cy3的荧光强度;(B)流式分析Cy3-siRNA的荧光值。
图8显示Western Blot检测siRNA纳米药物对肝癌细胞Huh7和SK-Hep-1中促癌基因ROC1的敲减效率,β-actin为内参;
图9为敲低癌基因ROC1,可以抑制肝癌细胞Huh7的增殖,促进其细胞凋亡,(A)该PDA基因药物对肝癌细胞Huh7的增殖抑制作用,(B)AnnexinV-FITC细胞流式凋亡图,(C)凋亡统计图;
图10为敲低癌基因ROC1,可以抑制肝癌细胞SK-Hep-1的增殖,促进其细胞凋亡,(A)该PDA基因药物对肝癌细胞SK-Hep-1的增殖抑制作用,(B)AnnexinV-FITC细胞流式凋亡图,(C)凋亡统计图;
图11显示负载siRNA的PDA基因纳米药物在敲低ROC1,抑制Neddylation Cullin1,促进凋亡因子ATF4的集聚;
图12显示control,NIR光照,blank NPs,blank NPs加NIR光照,PDA基因药物、以及PDA基因药物加NIR光照对Huh7裸鼠皮下瘤的抑制作用,(A)肿瘤体积的变化,(B)给药过程中老鼠体重的变化,取瘤体组织后(C)肿瘤的大小,(D)肿瘤的重量;
图13为(A)cleaved caspase-3、Ki-67及ROC1的免疫组化图,(B)免疫组化统计图。
图14显示小鼠活体成像观察基因纳米药物在心肝脾肺肾瘤等脏器的药物分布。
具体实施方式
下面结合附图和具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1--负载siRNA的PDA纳米药物的氨水制备法。
(1)首先用氨水的醇水溶液合成空白PDA纳米载体或siRNA负载的PDA纳米药物。先将3mL超纯水、1.2mL乙醇和15μL NH4OH在室温下搅拌30分钟。然后将625μl的PEI(1mg/ml,MW 100000)与625μl的siRNA(20μM)混匀孵育30分钟,混合物加入到(空白纳米载体不加)上述醇水的碱性溶液中。再加入20mg盐酸多巴胺,搅拌过夜。最后用离心法收集纳米药物,以10000rmp离心10分钟,取下层沉淀物。
(2)然后用EDC\NHS把FA修饰在纳米粒子的表面。首先将上述纳米药物(20mg)分散于1ml PBS缓冲液(10mM,pH=6.0)中。随后,在上述溶液中加入10mg FA。然后加入5mg的EDC和5mg的NHS,在搅拌条件下反应2小时。最后收集FA修饰的纳米药物,10000rmp离心10分钟,取下层沉淀物。
实施例2--负载siRNA的PDA纳米药物的Tris-HCl制备法。
先将720μl乙醇加入到4ml的Tris-HCl(10mM,pH=8.5)缓冲溶液中,在37℃下搅拌均匀。然后将625μl的PEI(1mg/ml,MW 100000)与625μl的siRNA(20μM)混匀孵育30分钟,混合物加入到(空白纳米载体不加)上述醇水的碱性溶液中。再加入20mg盐酸多巴胺,搅拌过夜。最后用离心法收集纳米药物,以10000rmp离心10分钟,收集下层沉淀物。
对于靶向分子FA的修饰,首先将上述纳米药物(20mg)分散于10ml PBS缓冲液(10mM,pH=6.0)中。随后,在上述溶液中加入10mg FA。然后加入5mg的EDC和5mg的NHS,在搅拌条件下反应2小时。最后收集FA修饰的纳米药物,10000rmp离心10分钟,收集下层沉淀物。
本发明以下试验例1-9使用透射电子显微镜(TEM)及动态光散射(DLS)等手段表征制备的PDA纳米载体,然后利用CCK-8法评价纳米载体及NIR光热治疗的细胞毒性。再通过体外实验验证纳米药物联合NIR光热治疗更能抑制肿瘤细胞的增殖,促进其凋亡。最后建立裸鼠皮下肿瘤模型进行抗肿瘤实验。
试验例1--siRNA纳米药物联合NIR光热疗法来治疗肝癌。
如图1所示,Neddylation在活化酶E1,结合酶E2以及连接酶E3的作用下被激活,促进肿瘤的发生发展。在肝癌病人组织中检测到高度活化的Neddylation,以及高表达的活化酶E1(NAE1,UBA3)、结合酶E2(UBC12)及连接酶E3(ROC1)等,如果敲低这些抗癌靶点,势必会抑制肝癌组织中Neddylation的活性,从而抑制肝癌的发生发展。申请人在本项目中主要利用siRNA负载的PDA基因纳米敲低促癌基因ROC1(E3),以探索该基因纳米药物联合NIR光热治疗对肝癌的抑制作用。如图2所示,肝癌病人分期越晚,ROC1的表达越高。而且ROC1高表达的肝癌病人的生存率较低。这些数据说明,ROC1的高表达与肝癌病人的预后不良成正相关。所以,敲低促癌基因ROC1,能抑制肝癌的发生发展。
为了提高基因药物的治疗效果,开发了具有刺激响应性和靶向分子修饰的有机纳米药物。在PDA纳米药物的制备方面,采用经典的Stober方法(盐酸多巴胺在碱性醇水溶液中还原为PDA),合成了可生物降解的PDA纳米药物。在反应过程中,混合溶液迅速从无色变为黑色,表明PDA纳米载体的形成。随后,在EDC和NHS存在下,通过氨基和羧基的共价偶联反应,将靶向分子FA修饰在PDA纳米药物表面。如图3所示,纳米药物的递送过程可以描述如下。首先,FA修饰的纳米药物被叶酸受体高表达的肿瘤细胞识别。纳米药物通过FA及其受体介导的内吞作用经核内体进入细胞质。溶酶体逃逸后,纳米药物被送到细胞质中。肿瘤细胞有氧糖酵解产生的酸性代谢物(乳酸等)在细胞内低pH条件下积累,从而刺激PDA纳米药物,释放抗肿瘤药物siRNA抑制癌细胞增殖。最后,PDA纳米药物联合其NIR光热治疗,具有更好的治疗效果。
试验例2--PDA纳米载体的表征。
利用透射电镜(TEM)和动态光散射(DLS)对其进行了表征。先将少量的PDA纳米载体悬浮液滴在湿润的培养皿中的封口膜上,将囊泡放置在碳涂层网格(300目)上3分钟,然后使用透射电镜(JEM-1230,JEOL)对网格进行分析。在DLS研究中,使用MalvernZetaSizer纳米ZS90粒度分析仪分析了纳米颗粒的粒度分布。
如图4A所示,TEM图像表明,PDA纳米载体具有球形且均匀的形貌。如图4B所示,DLS图像与TEM结果较为一致,大多数纳米粒子的尺寸约为330nm。
siRNA在PDA纳米药物中的包封率。根据siRNA(260nm)或者Cy3-siRNA(553nm)的标准曲线(吸光度与浓度),然后PDA基因纳米药物溶解于0.1M的NaOH溶液中,测得siRNA或者Cy3-siRNA的量,最后计算出siRNA或Cy3-siRNA在PDA纳米药物中的包封率。siRNA包封效率为72.3%,计算方法为纳米药物中siRNA包封量与加入siRNA总量之比。
试验例3--PDA纳米载体的生物相容性。
本发明选择天然黑色素PDA作为基本组成,以保证纳米载体具有优良的生物相容性。理论上,PDA纳米载体由于其可降解成高香草酸和二羟基杏仁酸,而具有良好的生物相容性。将QSG-7701、Huh7和SK-Hep-1细胞置于96孔板中,每孔2×103个细胞,4个复孔,培养过夜。不同试剂孵育72小时后,根据厂家说明书对细胞进行CCK-8检测。如图5所示,PDA纳米载体对肝正常细胞QSG-7701和肝癌Huh7以及SK-Hep-1细胞均无明显的细胞毒性,说明PDA纳米载体具有良好的生物相容性。另外,该专利也探讨了808激光对细胞生长的影响。如图5的最后一列所示,2W·cm-2的808激光5分钟对QSG-7701、Huh7和SK-Hep-1细胞的增殖几乎没有影响。
试验例4--Cy3-siRNA负载PDA纳米药物的智能释放,以及PDA纳米药物的NIR光热响应。
利用Cy3的光学信号,探索Cy3-siRNA纳米药物的智能化响应现象,其制备方法与siRNA一致。首先,将10mg Cy3-siRNA负载的PDA纳米药物溶解于10ml的1×PBS缓冲液(pH=7.4)中,分成10份,每份1ml,其中5个作为对照组(pH=7.4),另外5个作为对照加NIR光照组。同时,将10mg Cy3-siRNA负载的PDA纳米药物溶解于10ml 1×PBS缓冲液(pH=5.0)中,分成10份,每份1ml,其中5个作为pH(pH=5.0)释放组,另外5个作为pH(pH=5.0)释放加激光照射(808nm,2W·cm-2,5分钟)组。然后在预定的时间间隔离心沉淀不同组的4个样品,在553nm处用紫外分光光度计测定上清中Cy3-siRNA的吸光值,根据标注曲线计算出siRNA的含量。
大量研究表明,肿瘤组织的pH水平(6.5-6.9)普遍低于正常组织,这主要是由于肿瘤组织的有氧糖酵解(Warburg效应)和产生大量的酸性代谢物(乳酸等)。研究了在低pH水平(pH=5.0)下,在近红外激光照射和无近红外激光照射下,PDA纳米药物的pH释放和NIR光热响应。如图6A所示,释放曲线显示,对照组(pH=7.4的PBS缓冲液)的Cy3-siRNA释放量仅为19%。少量的siRNA泄漏是由于PDA纳米载体在正常情况下的相对稳定性造成的。而经过808激光处理(2W·cm-2,5分钟)后,PDA纳米药物的温度逐渐升高,导致Cy3-siRNA的释放量增加到27%,这可能是因为NIR激光照射导致的热胀释放效应。接着用pH=5.0的PBS缓冲液和pH=5.0的PBS缓冲液在NIR激光照射12小时内,DOX的释放量分别达到57%和78%。这种依赖pH的方式可能是由于PDA纳米载体的pH敏感性。外加NIR激光照射,PDA纳米药物的温度逐渐升高,导致Cy3-siRNA累积释放量显著增加,这也可能是热胀释放效应所致。此外,大多数siRNA在最初6小时内从PDA纳米药物中释放出来。这些结果证实了PDA纳米药物可以对低pH水平产生响应,并相应地触发药物的按需释放。同时,808激光治疗可加速PDA纳米药物中抗癌药物的释放。
然后,对PDA纳米药物的NIR光热响应进行了研究。808激光处理后PBS缓冲液、PDA纳米载体和负载siRNA的PDA纳米药物的曲线图及温度图(图6B)所示。用808激光处理后,PBS缓冲液的温度没有变化。而PDA纳米载体和负载siRNA的PDA纳米药物的温度随时间增加而达到饱和状态。此外,PDA纳米载体和负载siRNA的PDA纳米药物的温度曲线也有相似的趋势。这些结果说明,负载siRNA的PDA纳米药物具有利用NIR光热疗法治疗肝癌的能力。
试验例5--FA修饰的靶向给药作用,以及该纳米药物的转染效率。
为了分析FA的靶向作用,首先将肝正常细胞QSG-7701、肝癌细胞Huh7和SK-Hep-1分别接种于共聚焦培养皿,每孔2×105个细胞,培养24小时。然后,将上述细胞与0.2mg/ml负载Cy3-siRNA(1μg/ml)的FA修饰的PDA纳米药物孵育6小时。再用PBS洗涤,4%多聚甲醛固定30分钟,PBS洗涤后用0.5g/ml Hoechst 33258染色5分钟。最后,超纯水冲洗干燥后,在红色通道处用荧光显微镜(Leica)观察FA的靶向作用。或者收集细胞,在PE(红色)通道用流式细胞仪(BD)分析FA的靶向作用。
为了提高纳米药物的疗效和减少脱靶效应,将FA作为靶向分子修饰在PDA纳米药物表面。为验证FA的靶向作用,利用Cy3的光学信号,将Cy3-siRNA负载的FA修饰的PDA纳米药物与肝正常细胞QSG-7701、肝癌Huh7和SK-Hep-1细胞孵育。如图7A所示,荧光显微镜观察显示肝正常细胞QSG-7701周围有少量PDA纳米药物。QSG-7701细胞周围的红色可能是PDA纳米药物的非特异性吸附造成的,如流式细胞分析中QSG-7701细胞右移(红色,图7B)。相比之下,纳米药物在肝癌Huh7和SK-Hep-1细胞周围有大量的靶向吸附,说明FA在肿瘤细胞中具有良好的靶向给药作用。如图7B所示,流式细胞分析结果与荧光显微镜结果一致。这些数据表明,将FA作为靶向分子可以特异性地将抗癌药物递送到肿瘤细胞,而且根据肝癌Huh7及SK-Hep-1细胞的荧光显微镜图及流式细胞仪图可知,该基因纳米药物在肝癌细胞中的转染效率较高,基本在95%以上。
试验例6--siRNA负载的PDA纳米药物对肝癌细胞促癌基因ROC1的敲减效率。
根据该PDA基因纳米药物在肝癌细胞中高的转染效率,用Western blot检验体外细胞实验中该PDA基因纳米药物对肝癌细胞Huh7和SK-Hep-1中促癌基因ROC1的敲减效率。siRNA负载的FA修饰的PDA基因纳米药物,其中Cy3修饰的siRNA敲减促癌基因ROC1的序列是:5’-GACTTTCCCTGCTGTTACCTAATT-3’。然后,用Western blot检测该PDA基因纳米药物对促癌基因ROC1的敲减效率。先将1×106的肝癌细胞Huh7和SK-Hep-1接种于6孔板中,培养24小时。并用PDA基因纳米药物(含1μg/ml的siRNA)或含有随机NT序列的基因纳米药物与肝癌细胞Huh7及SK-Hep-1共同孵育72小时,然后用Western blot检测敲减效率。如图8所示,在肝癌细胞Huh7和SK-Hep-1中,该PDA纳米药物都能很好地敲减ROC1,其随机NT序列不能有效地敲减促癌基因ROC1。这些结果说明,该PDA基因纳米药物可以有效地敲低肝癌细胞中的促癌基因ROC1。
试验例7--siRNA负载的PDA纳米药物对肝癌细胞的体外抑制作用。
接着分析敲减促癌基因ROC1后,肝癌细胞的增殖和凋亡现象。在细胞增殖实验中,先将2×103个Huh7和SK-Hep-1细胞接种于96孔板中,培养24小时。并用负载siRNA的PDA纳米药物,负载siNT的PDA纳米药物,负载siRNA的PDA纳米药物联合NIR光热治疗72小时后,然后用CCK-8检测细胞的增殖情况。如图9A所示,敲低ROC1可以有效地抑制肝癌细胞Huh7的增殖,而随机NT序列对Huh7的增殖基本没有影响,PDA纳米药物联合NIR光热治疗,可以更好地抑制肝癌细胞的增殖。随后又验证了敲减ROC1后,肝癌细胞Huh7的凋亡现象。如图9B和9C的AnnexinV-FITC细胞流式凋亡图和凋亡统计分析所示,肝癌细胞Huh7通过该PDA纳米药物敲低促癌基因ROC1后,其凋亡现象明显增加,而NT序列对Huh7的凋亡基本没有影响,而PDA纳米药物联合NIR光热治疗,更能有效地促进肝癌细胞Huh7的凋亡。上述结果说明,该PDA基因纳米药物通过敲低促癌基因ROC1,可以抑制肝癌细胞Huh7的增殖,促进其凋亡。联合808激光照射的PDA纳米药物更能抑制Huh7的增殖,促进其凋亡。
图10A,10B和10C的肝癌细胞SK-Hep-1增殖实验和凋亡实验表明,该PDA基因纳米药物敲低促癌基因ROC1,可以抑制肝癌细胞SK-Hep-1的增殖,促进其凋亡,而随机NT序列不能影响肝癌细胞SK-Hep-1的增殖和凋亡。而PDA基因纳米药物联合NIR光热治疗,更能抑制肝癌细胞SK-Hep-1的增殖,促进其凋亡。808激光对肝癌细胞的抑制作用主要是由于热消融所致。这些结果说明,FA的靶向作用以及PDA纳米材料的pH响应,使得该PDA基因纳米药物具有良好的抗肝癌作用,联合NIR光热治疗,该PDA基因纳米药物具有更为优良的抗肝癌作用。
试验例8--siRNA负载的PDA纳米药物抑制肝癌的机制研究。
本项目通过敲低Neddylation的促癌基因ROC1(E3),抑制Neddylation通路,达到抑制肝癌发生发展的目的。如图11所示,在肝癌细胞Huh7以及SK-Hep-1都可以观察到,基因纳米药物通过敲低ROC1,显著抑制ROC1的底物Cul1的Neddylation修饰,而对Cul5的Neddylation修饰影响较小,这是因为Cul5是ROC2(Rbx2/SAG,E3)的底物。同时,敲低ROC1,诱导凋亡因子ATF4的集聚,促进了肿瘤细胞的凋亡。而纳米药物携带的随机序列没有这样的作用。
试验例9--siRNA负载的PDA纳米药物联合NIR光热照射的体内抗肿瘤作用。
购自中国上海斯莱克实验动物有限公司的6周龄雄性裸鼠(约20g),随机分为6组(每组n=6),在每只小鼠左侧腋窝皮下注射1×106的Huh7细胞。当肿瘤体积达到大约50mm3,每隔一天小鼠腹腔注射PBS(100μl),PBS加NIR激光照射(2W·cm-2,5分钟),PDA纳米载体(-2mg/kg),纳米载体加激光照射,siRNA纳米药物(含有siRNA 0.3mg/kg),siRNA纳米药物加激光照射,当最大肿瘤体积达到约1000mm3时,处死所有小鼠,测量肿瘤体积和重量。另外,用福尔马林固定肿瘤组织进行免疫组化分析。
如图12B所示,治疗期间各组均未观察到小鼠死亡或体重显著下降,说明治疗对荷瘤小鼠没有产生严重的毒副作用。如图12A所示,NIR激光照射以及PDA纳米载体与对照组有相似的趋势,说明激光照射对荷瘤小鼠没有明显的影响,PDA纳米载体具有良好的生物相容性。而纳米载体加NIR光热治疗具有显著的抑瘤的作用,基因纳米药物组表现出比纳米载体加NIR光热治疗略好的抑制作用,说明该纳米药物具有光热治疗的作用,而且基因治疗可以通过抑制促癌基因ROC1抑制肝癌的生长。基因纳米药物加NIR光热治疗组几乎抑制了肝癌的发生发展,表明该基因纳米药物联合光热治疗可以达到更为有效地抑制肝癌生长的目的。肿瘤照片(图12C)和肿瘤重量(图12D)与肿瘤体积的结果一致,这些结果表明,近红外激光照射的PDA纳米药物对肿瘤的抑制能力最强,这是由于靶向递送的PDA纳米药物在肿瘤微环境的低pH水平刺激释放所致。此外,激光辐照的热消融也提高了PDA纳米药物的治疗效果。
免疫组化分析,各组肿瘤用5ml福尔马林固定过夜,乙醇脱水,石蜡包埋,切片(5μm)。载玻片在二甲苯和乙醇中脱蜡,再在水中水化。然后在PBS缓冲液(pH=6.0)中微波加热30分钟进行抗原提取。再将载玻片在3%过氧化氢中猝灭,以阻断内源性过氧化物酶活性,用TBST缓冲液洗涤。最后,一抗4℃孵育过夜,按照SuperPictureTM聚合物检测试剂盒(LifeTechnologies)说明书进行,使用cleaved caspase 3及Ki-67(Abcam)抗体。如图13A和13B所示,NIR光照组以及PDA纳米载体组凋亡因子(cleaved-caspase-3)以及生长因子(Ki-67)的阳染与对照组相似,说明NIR光照以及PDA纳米载体优良的生物相容性。PDA纳米载体的NIR光照组,以及PDA纳米药物组具有更多的凋亡因子(cleaved-caspase-3)以及更少的生长因子(Ki-67)的阳染,说明该纳米载体具有光热治疗的作用,基因纳米药物可以通过敲低促癌基因ROC1,抑制肿瘤的发生发展。基因纳米药物加NIR光热治疗组,具有最多的凋亡因子(cleaved-caspase-3)以及最少的生长因子(Ki-67),因此更能抑制肝癌的生长。
利用Cy3-siRNA的荧光信号,通过体内成像系统监测free Cy3-siRNA,Cy3-siRNA负载的PDA纳米药物,以及叶酸修饰的Cy3-siRNA负载的PDA纳米药物的体内药物分布。如图14所示,在尾静脉注射24小时后,free Cy3-siRNA以及Cy3-siRNA负载的PDA纳米药物在肿瘤内看不到明显的Cy3-siRNA荧光,这是由于活体成像仪的低灵敏度所致。相比之下,经FA修饰的基因纳米药物在肿瘤部位可以观察到明显的Cy3-siRNA荧光。而在肝脏组织中,未经FA修饰的基因纳米药物的荧光强度较强,表现出明显的脱靶效应和毒副作用。这些结果表明,靶向修饰的PDA纳米药物可以提高治疗效果,减少脱靶和毒副作用。
Claims (5)
1.一种负载siRNA/PEI的PDA纳米药物的制备方法,其特征是,包括如下步骤:
(1)分子量MW=200-100000的PEI与siRNA反应10-120分钟,制得siRNA/PEI混合物,其中siRNA与PEI的质量比为1:1-1:10;配制氨水或Tris-HCl的醇水溶液,醇水溶液中乙醇和水的体积比为1:1-1:10;在氨水或Tris-HCl的醇水溶液中加入siRNA/PEI混合物和盐酸多巴胺,siRNA与盐酸多巴胺的质量比为1:1-1:1000,氨水或Tris-HCI与盐酸多巴胺的摩尔比为1:1-1:50,搅拌,离心收集PDA纳米药物;
(2)靶向分子的修饰:将PDA纳米药物分散于PBS缓冲液中,用EDC/NHS通过氨基和羧基的耦合反应,结合带羧基的靶向分子叶酸,EDC/NHS与盐酸多巴胺的摩尔比为1:1-1:10,叶酸与盐酸多巴胺的摩尔比为1:1-1:50,离心收集靶向分子修饰的PDA纳米药物,制得负载siRNA/PEI的PDA纳米药物。
2.根据权利要求1所述的负载siRNA/PEI的PDA纳米药物的制备方法,其特征是,步骤(2)中,耦合反应的温度为10-40℃,反应时间为1-48小时。
3.根据权利要求1所述的负载siRNA/PEI的PDA纳米药物的制备方法,其特征是,步骤(1)、(2)中,离心转速为3000rpm-15000rpm,离心时间为2-30分钟。
4.权利要求1或2或3所述的制备方法制得的负载siRNA/PEI的PDA纳米药物。
5.权利要求1或2或3所述的制备方法制得的负载siRNA/PEI的PDA纳米药物在制备治疗肝癌的药物中的用途。
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