CN113230400A - 一种基于表观遗传调控的金纳米团簇放疗增敏组装体及其制备和应用 - Google Patents

一种基于表观遗传调控的金纳米团簇放疗增敏组装体及其制备和应用 Download PDF

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CN113230400A
CN113230400A CN202110263586.0A CN202110263586A CN113230400A CN 113230400 A CN113230400 A CN 113230400A CN 202110263586 A CN202110263586 A CN 202110263586A CN 113230400 A CN113230400 A CN 113230400A
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崔大祥
王立蕊
张春雷
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Shanghai Jiaotong University
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Abstract

本发明涉及一种基于表观遗传调控的金纳米团簇放疗增敏组装体及其制备和应用，该制备方法包括以下步骤：(1)取以谷胱甘肽为巯基配体的金纳米团簇，加入水与DMSO的混合溶液，充分重悬形成金纳米团簇溶液；(2)将聚烯丙基胺盐酸盐溶于水中，配成PAH溶液；(3)将SAHA溶于DMSO中，得到SAHA溶液；(4)将SAHA溶液加入金纳米团簇溶液，充分混合，再滴加PAH溶液，搅拌反应，所得反应溶液透析纯化，即得到目的产物。本发明的制备方法操作简便，条件温和，所制备的组装体可以利用金纳米团簇和表观遗传抑制剂的协同放疗增敏效应增加肿瘤细胞对辐照的敏感性，在肿瘤的放射治疗中具有广阔的应用前景。

Description

一种基于表观遗传调控的金纳米团簇放疗增敏组装体及其制 备和应用
技术领域
本发明属于纳米药物制备技术领域，涉及一种基于表观遗传调控的金纳米团簇放疗增敏组装体及其制备和应用。
背景技术
放射治疗与手术、化疗是治疗癌症的最有效的三大手段，超过50％的癌症患 者在于肿瘤的斗争中需要接受放射治疗。然而，目前临床上患者在接受放射治疗的 过程中，将会受到较高强度的射线辐射，导致在杀伤肿瘤的同时，对肿瘤周围的正 常组织也会产生不可避免的伤害。因此，研制高效的放疗增敏剂、降低所需辐射剂 量、在杀伤肿瘤的同时降低对正常组织的损伤是当前放射治疗策略面临的一大挑 战，亟待解决。
随着纳米技术的不断应用，越来越多的研究发现纳米颗粒具有增强的渗透性和保留(EPR)效应，这一特性有利于纳米药物在肿瘤组织的聚集，对于肿瘤的治疗 具有重大意义。因此，金属纳米颗粒，尤其是具有高原子序数和质量能量系数的纳 米颗粒，具有优异的放疗增敏特性，可以提高治疗效率并减轻副作用。
在众多的金属纳米颗粒之中，以谷胱甘肽(GSH)为配体的金纳米团簇(GNCs) 以其超小的尺寸、稳定的结构、优异的水溶性、良好的生物相容性以及较快的组织 清除速度，成为生物医学应用中一种很有前途的纳米材料。金纳米团簇在肿瘤诊断 和放疗中是一种良好的工具。此外，GNCs具有优异的自组装性能，主要是基于其 表面的负电荷与阳离子或阳性高分子表面的正电荷之间的静电相互作用所驱动，这 种组装体可用于癌症的多模态诊疗。除了金属纳米颗粒作为放疗增敏剂外，许多临 床使用的抗癌药也具有放疗增敏的效果。例如，组蛋白去乙酰化酶的抑制剂 Vorinostat(SAHA)可通过表观遗传调控促进DNA损伤并抑制损伤的DNA修复， 提高细胞对放疗的敏感性。然而，目前将具有放疗增敏性的表观遗传抑制剂和金属 纳米粒子相结合的纳米级高效放疗增敏剂还未有研究报道。
发明内容
本发明的目的就是为了提供一种基于表观遗传调控的金纳米团簇放疗增敏组 装体及其制备和应用，所制备的金纳米团簇放疗增敏组装体能够很好地被胃癌细胞 内吞，协同诱导胃癌细胞的DNA损伤，抑制小鼠移植瘤的生长，同时具有良好的 生物安全性。因此，该纳米组装体在胃癌等肿瘤的放射治疗中具有广阔的应用前景。
本发明的目的可以通过以下技术方案来实现：
本发明的技术方案之一提供了一种基于表观遗传调控的金纳米团簇放疗增敏 组装体的制备方法，其特征在于，包括以下步骤：
(1)取以谷胱甘肽为巯基配体的金纳米团簇，加入水与DMSO的混合溶液， 充分重悬形成金纳米团簇溶液；
(2)将聚烯丙基胺盐酸盐(PAH)溶于水中，配成PAH溶液；
(3)将SAHA溶于DMSO中，得到SAHA溶液；
(4)将SAHA溶液加入金纳米团簇溶液，充分混合，再滴加PAH溶液，搅 拌反应，所得反应溶液透析纯化，即得到目的产物。
进一步的，步骤(1)中，DMSO与水的体积比为4:1。
进一步的，步骤(4)中，SAHA溶液与金纳米团簇溶液在室温下混合，混合 时间为10min。
进一步的，步骤(4)中，金纳米团簇溶液、SAHA溶液和PAH溶液的添加量 满足：金纳米团簇、聚烯丙基胺盐酸盐和SAHA的摩尔比为180:35:1。
进一步的，步骤(4)中，搅拌反应在室温下进行，反应时间为2.5h。
进一步的，步骤(4)中，透析纯化在纯水中进行，纯化时间为24-48h，且每 6h置换一次纯水。
进一步的，步骤(4)中，透析纯化采用截留分子量为15kDa的装置进行透析。
进一步的，步骤(1)中，所述的金纳米团簇中所采用的谷胱甘肽为手性L-型 谷胱甘肽，且其中，谷胱甘肽和金原子的摩尔比为18:22。
与一般传统方法相比，首先，本发明中公开的方法简单，条件温和，采用一锅 法即可制备出形貌均一的新型纳米组装体；其次，本发明中采用具有良好生物安全 性的以谷胱甘肽为配体的金纳米团簇和FDA批准的临床药物SAHA，加入带正电 荷的PAH后自组装生成直径在30-80nm的颗粒，可以有效增加细胞对金纳米团簇 和小分子药物的摄取；最后，本发明公开的方法制备的纳米组装体可以利用金纳米 团簇和表观遗传抑制剂的协同放疗增敏作用，进一步达到治疗肿瘤的效果。
本发明中，对各原料试剂的添加量、处理工艺条件等均进行了限定，本发明主 要的反应与后处理主要是在室温下进行，GNCs表面的负电荷以及PAH表面的正 电荷具有较强的静电吸引作用，在室温下水溶液中即可进行，故实验安全简单，可 操作性强。在本发明中涉及到GNCs和SAHA充分混合的操作步骤，目的是为了 使GNCs和SAHA在DMSO:水溶液中均匀分散。在涉及自组装的步骤时，需要逐 滴滴加PAH的原因是防止大量的PAH和金纳米团簇发生络合聚集，影响后续的自 组装和自组装过程中SAHA的负载，故选用缓慢滴加的方法。
本发明的技术方案之二提供了一种基于表观遗传调控的金纳米团簇放疗增敏 组装体，其采用如上所述的制备方法制备得到。
本发明的技术方案之三提供了一种基于表观遗传调控的金纳米团簇放疗增敏 组装体在制备用于肿瘤放射治疗的药物中的应用。
优选的，适合的放射治疗的肿瘤为胃癌。
本发明以金纳米团簇、PAH和小分子药物SAHA为模块，通过PAH正电荷和 金纳米团簇所带负电荷之间的静电吸附作用将带小分子药物SAHA包裹于其中， 进一步自组装成为具有更大粒径在80-150nm的纳米颗粒。该纳米颗粒可以联合基 于表观遗传调控的小分子药物SAHA和金纳米团簇的放疗增敏效果，协同促进肿 瘤细胞对X射线的敏感性，引起细胞死亡。
与现有技术相比，本发明具有以下优点：
(1)所述的纳米组装体的制备条件温和，方法简单；
(2)采用原料小分子药物为FDA批准的临床用药；
(3)制备的纳米探针具有良好的协同放疗增敏效果；
(4)制备的纳米组装体具有良好的生物安全性。
附图说明
图1为纳米组装体的TEM表征图；
图2是纳米组装体被胃癌MGC-803细胞摄取的细胞TEM图；
图3是制备的纳米组装体的细胞毒性实验结果；
图4是制备的纳米组装体对胃癌细胞DNA损伤影响的实验结果；
图5是制备的纳米组装体对裸鼠结果。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。本实施例以本发明技术方案为前提进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范 围不限于下述的实施例。
以下各实施例中，聚烯丙基胺盐酸盐(即PAH)购于侨怡生物科技(上海) 有限公司，品牌为Macklin麦克林,CAS号为71550-12-4.其余如无特别说明的原 料试剂或处理技术，均为本领域的常规市售原料或常规处理技术。
实施例1：
金纳米团簇的制备和纯化：
将新鲜配制HAuCl4(2mL，0.05M)和GSH(2.5mL，0.1M)溶液，加入 90.5mL去离子水中，快速冰浴搅拌3分钟。加入5mL预冷的四丁基化铵(TBAB) 溶液，剧烈搅拌5分钟，静置1小时。用1M盐酸将上述溶液的pH调节至～3.0， 冰浴12小时，采用离心的方式去除不溶的Au(I)-SG复合物沉淀，得到金纳米团簇 的上清液。加入2倍体积的甲醇和氯化钠溶液(20mM)沉淀纯化金纳米团簇，真 空冷冻干燥，得到金纳米团簇，置于4℃备用。
实施例2：
负载SAHA的金纳米团簇放疗增敏组装体的制备，请再参见图1所示：
将上述实施例1制备的金纳米团簇按照50μg gold/mL溶于去离子水：DMSO (体积比为4:1)溶液中，将1mg SAHA溶解于0.1mL的DMSO中，将SAHA 溶液加入金纳米团簇溶液中，搅拌5分钟。将PAH按照2mg/mL浓度配制成水溶 液，逐滴PAH溶液滴加至金纳米团簇和SAHA的混合溶液中，室温搅拌2.5小时， 转速为1000转/分钟，以上满足金纳米团簇、聚烯丙基胺盐酸盐和SAHA的摩尔 比为180:35:1。此后，将上述溶液在纯水中透析24-48小时，期间每6小时置换一 次纯水。最终制备得到负载表观遗传抑制剂的金纳米团簇组装体，即为 GNCs-PAH/SAHA纳米探针。
经由实施例1-实施例2即可制得最后的负载表观遗传抑制剂SAHA的金纳米 团簇组装体。
图1是实施例2所制备的GNCs-PAH/SAHA组装体的TEM表征图，从图中可 以看出，SAHA负载的金纳米团簇组装体呈现的是球形结构，它由金团簇表面的负 电荷和PAH表面的正电荷相互吸引并在组装的过程中负载SAHA形成的较大的纳 米颗粒，在水溶液中呈单分散状态且大小均一。
实施例3：
GNCs-PAH/SAHA纳米颗粒被细胞内吞实验：
将MGC-803细胞接种于25cm2培养瓶中，当细胞贴壁其密度涨至～80％后， 弃掉培养基，加入含有实施例2所制备的GNCs-PAH/SAHA纳米颗粒的DMEM培 养基，继续培养4小时。弃掉培养液，用胰酶消化收集细胞，并用PBS洗2遍， 将细胞固定于2.5％(wt/vol)的戊二醛溶液中，过夜。离心收集细胞，用PBS洗涤， 得到纯细胞颗粒。然后用1％四氧化锇固定2小时，然后依次用乙醇(25、50、75、 95、100％)和丙酮处理脱水。接下来，将细胞转移到环氧丙烷中并嵌入环氧树脂 中，制作超薄切片并用乙酸铀酰染色，然后用柠檬酸铅染色。最后，将超薄切片转 移到铜栅上，用120kv透射电镜进行观察。
图2显示：GNCs-PAH/SAHA纳米颗粒与MGC-803细胞孵育后，存在于细胞 质基质中，说明GNCs-PAH/SAHA纳米颗粒可以被胃癌细胞内吞。
实施例4：
为了证实纳米颗粒的体外放疗增敏效果，MGC-803细胞以细胞密度为5×103个细胞/孔种植于96孔板内，每孔100μL，准备有5个复孔，并培养过夜。次日， 弃去上清，分别加入含有不同浓度的纳米颗粒与对应浓度的KGN，以加入PBS作 为空白对照，经共同孵育6小时后，弃上清，用PBS洗涤一次，然后将对细胞进 行4Gy剂量的X-射线辐照，并继续培养24小时。最后用CCK-8试剂盒对细胞活 性进行检测。
图3结果表明：与对照组相比，加入实施例2所制备的GNCs-PAH/SAHA纳 米颗粒处理组具有显著降低的细胞活性，说明该纳米组装体具有良好的放疗增敏效 果。
实施例5：
在X-射线辐照下，GNCs-PAH/SAHA纳米颗粒诱导MGC-803细胞发生DNA 损伤的能力：
将MGC-803细胞种于四室玻皿中(每孔7×104个细胞/mL)，孵育过夜。然 后去除细胞培养液，加入含有实施例2所制备的GNCs-PAH/SAHA纳米颗粒的 DMEM培养液，孵育12小时。弃掉培养液，用PBS洗涤，用X-射线辐照(4Gy), 继续培养24小时，。用4％多聚甲醛室温下固定细胞20分钟后，再用PBS洗涤 4次，加入含0.05％Triton的PBS渗透5分钟，PBS洗脱5次，然后用5％BSA室 温封闭2小时。接着用rH2AX抗体的稀释液于4℃孵育过夜，弃去一抗，PBS洗 涤5次，加入荧光标记的二抗，室温避光孵育1小时，再用2μg/mL的Hoechst 33342 复染细胞核。
图4结果显示：与未处理的细胞相比，免疫荧光染色显示出GNCs-PAH/SAHA 纳米颗粒处理的细胞在X-射线辐照后在其细胞核中表现出明显增多的rH2AX焦 点，表明有GNCs-PAH/SAHA纳米颗粒可以显著促进X-射线对MGC-803细胞的 DNA损伤。
实施例6：
评价GNCs-PAH/SAHA纳米颗粒的抑瘤实验。
将MGC-803细胞皮下种植到裸鼠右后侧，建立肿瘤模型，随机将裸鼠分为4 组：对照组、GNCs组、SAHA组和纳米颗粒组(即含有实施例2所制备的 GNCs-PAH/SAHA纳米颗粒的实验组)。待肿瘤生长至～100mm3时，对肿瘤进行 瘤内注射，24h后进行X射线放射治疗(6Gy剂量)。每隔三天测量一次肿瘤体 积，15天后治疗结束，记录肿瘤的大小变化情况。
说明：GNCs组为参照实施例2，但不引入SAHA的实验组；SAHA组为参照 实施例2，但不引入金纳米团簇的实验组。
另外，本发明还通过实验发现，当不引入PAH时，GNCs和SAHA不能成功 组装成纳米颗粒，处于游离状态。
图5显示：纳米颗粒处理组在X射线辐照下具有最显著的抑瘤效果，说明其 具有优良的放疗增敏效果。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此 说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限 于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改 进和修改都应该在本发明的保护范围之内。

Claims (10)

1.一种基于表观遗传调控的金纳米团簇放疗增敏组装体的制备方法，其特征在于，包括以下步骤：
(1)取以谷胱甘肽为巯基配体的金纳米团簇，加入水与DMSO的混合溶液，充分重悬形成金纳米团簇溶液；
(2)将聚烯丙基胺盐酸盐溶于水中，配成PAH溶液；
(3)将SAHA溶于DMSO中，得到SAHA溶液；
(4)将SAHA溶液加入金纳米团簇溶液，充分混合，再滴加PAH溶液，搅拌反应，所得反应溶液透析纯化，即得到目的产物。
2.根据权利要求1所述的一种基于表观遗传调控的金纳米团簇放疗增敏组装体的制备方法，其特征在于，步骤(1)中，DMSO与水的体积比为4:1。
3.根据权利要求1所述的一种基于表观遗传调控的金纳米团簇放疗增敏组装体的制备方法，其特征在于，步骤(4)中，SAHA溶液与金纳米团簇溶液在室温下混合，混合时间为10min。
4.根据权利要求1所述的一种基于表观遗传调控的金纳米团簇放疗增敏组装体的制备方法，其特征在于，步骤(4)中，金纳米团簇溶液、SAHA溶液和PAH溶液的添加量满足：金纳米团簇、聚烯丙基胺盐酸盐和SAHA的摩尔比为180:35:1。
5.根据权利要求1所述的一种基于表观遗传调控的金纳米团簇放疗增敏组装体的制备方法，其特征在于，步骤(4)中，搅拌反应在室温下进行，反应时间为2.5h。
6.根据权利要求1所述的一种基于表观遗传调控的金纳米团簇放疗增敏组装体的制备方法，其特征在于，步骤(4)中，透析纯化在纯水中进行，纯化时间为24-48h，且每6h置换一次纯水。
7.根据权利要求1所述的一种基于表观遗传调控的金纳米团簇放疗增敏组装体的制备方法，其特征在于，步骤(4)中，透析纯化采用截留分子量为15kDa的装置进行透析。
8.根据权利要求1所述的一种基于表观遗传调控的金纳米团簇放疗增敏组装体的制备方法，其特征在于，步骤(1)中，所述的金纳米团簇中所采用的谷胱甘肽为手性L-型谷胱甘肽，且其中，谷胱甘肽和金原子的摩尔比为18:22。
9.一种基于表观遗传调控的金纳米团簇放疗增敏组装体，其采用如权利要求1-8任一所述的制备方法制备得到。
10.如权利要求9所述的一种基于表观遗传调控的金纳米团簇放疗增敏组装体在制备用于肿瘤放射治疗的药物中的应用。
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