CN111920947B - 一种花菁染料介导的核酸/抗癌药物复合物的制备方法 - Google Patents
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Abstract
本发明公开了一种花菁染料介导的核酸/抗癌药物复合物的制备方法，本发明以花菁染料修饰的功能核酸和抗癌化疗药物为原料，使其在水溶液中通过分子静电引力、质子的转移和疏水作用，自组装形成核酸/抗癌药物复合物。本发明制备功能核酸/抗癌药物复合物的方法简单、绿色、温和且成本低廉，其中抗癌化疗药物与功能核酸的协同作用可实现化疗和免疫治疗的良性结合，达到治疗癌症的目的。因此，本发明有望为制备高效的抗癌功能核酸药物提供理论和实验的技术支持。
Description
技术领域
本发明属于生物医药领域，具体涉及一种花菁染料介导的核酸/抗癌药物复合物的制备方法。
背景技术
功能核酸，包括siRNA，mRNA和反义核酸，已经广泛用于癌症的诊断和治疗。然而，单一手段的核酸治疗极易受到核酸酶降解和低的转染效率的限制，严重制约了功能核酸的应用。功能核酸和其他治疗手段（包括，放疗、化疗、光动力治疗、光热治疗）结合可以最大化癌症治疗的效果。化疗作为临床癌症治疗的标准手段，其与功能核酸的协同治疗展现出较大的应用潜力。然而，化疗药物多数为小分子，由于其与核酸物理化学性质具有较大差异，很难实现核酸和化疗药物直接装载在同一个纳米颗粒中。因此，直接实现功能核酸和化疗药的组装成为制备核酸药物的关键手段。
花菁染料作为常见的染料分子，广泛用于核酸修饰，并可进一步用于核酸的定量分析、荧光定位。但是，对于花菁染料的物理化学性质以及组装性能很少有报道。众所周知，花菁染料属于芳香类染料，具有苯环结构。因此，花菁染料具有很强的π共轭体系和疏水作用。此外，花菁染料侧链端具有磺酸基团，使其表现出较强的负电性。理论上，花菁染料极易与正电荷、π共轭的药物结合。目前，临床所用的抗癌药多属于芳香化合物，具有很好的共轭性能。此外，亲水性的化疗药物很多表现出正电性，利于被细胞吞噬。因此，将带正电的抗癌药和花菁类修饰的功能核酸混合时，由于静电引力、π-π堆积等相互作用，二者很容易形成核酸/抗癌药物的纳米颗粒，对于开发制备具有不同功能的核酸药物具有重要的科学意义。
发明内容
本发明的目的在于提供一种花菁染料介导的核酸/抗癌药物复合物的制备方法，其采用一种简单的方法设计出一种化疗和免疫治疗协同作用的复合药物，可以实现有效的抗肿瘤治疗。
为实现上述目的，本发明采用如下技术方案：
一种花菁染料介导的核酸/抗癌药物复合物的制备方法，其是将花菁染料修饰的功能核酸和抗癌化疗药物在水溶液中充分混合均匀，以利用质子偏移、静电引力和亲疏水变化自组装成尺寸均匀、负载率高的新型功能核酸/抗癌药物复合物；其具体包括以下步骤：
（1）花菁染料修饰的功能核酸原液的制备：将花菁染料修饰的功能核酸溶解在无菌水中，室温、避光条件下吹打均匀，形成均匀的花菁染料修饰的功能核酸原液，其浓度为100 μM；
（2）药物原液的制备：将抗癌化疗药物溶解在水、二甲基亚砜等溶剂中，室温、避光条件下混合均匀，得到药物原液，其浓度为10 mM；
（3）核酸/抗癌药物复合物的制备：将花菁染料修饰的功能核酸原液与药物原液混合均匀，置于金属浴中，95 ℃反应1 h，然后冷却至室温，再将产物离心，二次水洗两次，制备得到所述核酸/抗癌药物复合物；离心的转速为8000 rpm，时间为5 min。
所述花菁染料为常规已知的任意花菁染料中的一种，如Cy3、Cy3.5、Cy5、Cy5.5、Cy7和Cy7.5等。
所述功能核酸为常规已知的任意功能核酸中的一种，如CPG、siRNA、mRNA、反义核酸、DNA等。
所述花菁染料修饰的功能核酸是将花菁染料通过酰胺缩合反应修饰在功能核酸的一端，每条功能核酸修饰一个花菁染料分子。
所述抗癌化疗药物为盐酸佐柔比星、盐酸阿霉素或阿霉素的结构类似物及衍生物。
所用花菁染料修饰的功能核酸与抗癌化疗药物的摩尔比为1:30。
本发明的有益效果在于：
本发明公开了一种花菁染料介导的核酸/抗癌药物复合物的制备方法，该方法简单、原料易得，具有广泛的通用性。其中所用抗癌化疗药物，如盐酸阿霉素可以有效的抑制DNA的转录，诱导细胞凋亡，并诱导机体的免疫原性死亡，激活免疫反应。而功能核酸，如CPG可以促进抗原呈递细胞的成熟，增强抗原呈递的能力，进一步增强机体的免疫反应。
本发明采用了一种简单的方法设计出一种化疗和免疫治疗协同作用的核酸/抗癌药物复合物，其可以实现有效的抗肿瘤治疗，且其作为一种通用方法，可为核酸药物的制备提供良好的参考。
附图说明
图1为实施例制备Cy5-CPG/DOX的反应流程图。
图2为实施例制备的Cy5-CPG/DOX的透射电镜图（a）、扫描电镜（b）、粒径分布图（c）和电位变化图（d）。
图3为实施例制备的Cy5-CPG/DOX的性能试验测试结果图，其中（a）为Cy5-CPG/DOX的循环稳定性试验结果，（b）为Cy5-CPG/DOX在不同环境下的释放情况图，（c）为Cy5-CPG/DOX以及Cy5-CPG、DOX诱导细胞凋亡的情况图，（d）为细胞对Cy5-CPG/DOX以及Cy5-CPG、DOX的内吞率情况图。
图4为实施例制备的Cy5-CPG/DOX诱导细胞免疫反应的情况图。
具体实施方式
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是本发明不仅限于此。
实施例
如图1，以花菁染料修饰的功能核酸Cy5-CPG和盐酸阿霉素（DOX）组装为例，制备花菁染料介导的核酸/抗癌药物复合物；其具体制备步骤如下：
（1）将10 OD 的Cy5-CPG（购买于上海生物工程有限公司）溶解在527 μL的无菌水中，避光、室温条件下吹打，使核酸分散均匀，形成100 μM的Cy5-CPG原液，避光-20℃保存；
（2）室温避光条件下，将57.9 mg的盐酸阿霉素（DOX，购买于sigam公司）分散在10mL的二次水中，充分分散均匀，得到10 mM的DOX原液，避光保存；
（3）将15 μL的Cy5-CPG原液，5 μL的DOX原液（Cy5-CPG与DOX的摩尔比为1:30）和300μL的二次水充分混合，然后置于金属浴，95 ℃反应60 min，然后冷却至室温，将样品取出，再于8000 rpm离心5 min，二次水洗两次，得到Cy5-CPG/DOX。
图2为制备得到的Cy5-CPG/DOX的透射电镜（a）、扫描电镜图（b）、粒径分布图（c）及电位变化图（d）。从图中可以观察到，Cy5-CPG和盐酸阿霉素复合形成的纳米颗粒尺寸均匀，其粒径集中分布在200 nm，纳米颗粒的表面电荷为-32.3 mV。
性能试验
1. 取500 μg/mL的Cy5-CPG/DOX样品，分别与1 mL的PBS、5%血清（FBS）和细胞裂解液混合，在37℃孵育不同时间，并每隔一定时间取反应液，8000 rpm离心3 min后，取上清100 μl，用酶标仪测定593 nm处的荧光峰。
2. 以乳腺癌细胞系4T1为验证模型。将4T1细胞接种在6孔板中，孵育24 h，用PBS清洗三次。将细胞裂解，离心取上清各1 mL。然后用PBS缓冲液和H2O2调节其pH值，得到pH为7.4和5.0的细胞裂解物混合溶液。分别加入500 μg/mL的Cy5-CPG/DOX，孵育不同时间，并每隔一定时间取反应液，8000 rpm离心3 min后，取上清100 μL，用酶标仪测定593 nm处的荧光峰。
3. 以乳腺癌细胞系4T1为验证模型。将4T1细胞接种在96孔板中，孵育24 h，用PBS清洗三次。分别加入含有不同浓度（0、50、100、150、200、250、300μg/ml）各化合物（Cy5-CPG、DOX、Cy5-CPG/DOX）的培养基100 μL，孵育24 h。然后取出上清，用PBS清洗三次，加入含有CCK-8（Cell Counting Kit-8，碧云天）的培养基100 μL，孵育1h，用酶标仪测定450 nm的吸收值。
4. 以乳腺癌细胞系4T1为验证模型。将4T1细胞接种在6孔板中，孵育24 h，用PBS清洗三次。分别加入含有300μg/ml的各化合物（Cy5-CPG、DOX、Cy5-CPG/DOX）的培养基1 mL，孵育12 h。然后用PBS清洗细胞，离心收集细胞。将细胞裂解，离心取上清测定593 nm处的荧光峰。
5. 以乳腺癌细胞系4T1为验证模型。将4T1细胞接种在荧光板中，孵育24 h，用PBS清洗三次。然后，分别加入含有300 μg/ml Cy5-CPG/DOX 的培养基1 mL，共同孵育6 h。细胞用PBS清洗三次，然后加入4%组织固定液，固定10 min。再加入含有钙网蛋白的抗体孵育30min，进一步用FITC标记的IgG孵育60 min。用共聚焦显微镜测定细胞由于免疫原性死亡而暴露的钙网蛋白的表达。
图3为性能试验测试结果图，其中（a）为Cy5-CPG/DOX体外循环稳定性的试验结果，由图中可见，由于Cy5-CPG/DOX具有较好的结合力，因此在PBS中可以保存较长时间；而在血清中，虽然Cy5-CPG/DOX表现出一定的DOX释放性能，但是保留时间较长，因此具有较好的血液循环稳定性；在细胞裂解物混合溶液中，Cy5-CPG/DOX释放出较多的DOX，说明纳米颗粒可以在细胞中释放，释放的药物可以更好的诱导细胞凋亡。
（b）为Cy5-CPG/DOX在细胞内不同环境下的释放情况图。由图中可以看出，在pH=5的肿瘤微环境条件下，Cy5-CPG/DOX可以更好的释放，表现出一定的弱酸响应性。
（c）和（d）分别为Cy5-CPG/DOX以及Cy5-CPG、DOX诱导细胞凋亡和细胞内吞的情况图。图中可以观察到，Cy5-CPG/DOX所形成的纳米颗粒具有较好的内吞作用，并展现出最优的细胞杀伤效果。
图4为Cy5-CPG/DOX诱导细胞免疫反应的情况图。图中可以观察到，DOX可以诱导细胞凋亡，引起免疫原性死亡，因此引起钙网蛋白的泄露。
由上述试验可见，所制备的Cy5-CPG/DOX可以较好的被细胞内吞，在肿瘤细胞微环境中释放DOX诱导细胞的凋亡，凋亡的细胞可以作为抗体引起免疫原性死亡。CPG作为抗原呈递细胞的刺激佐剂，可以激活抗原呈递细胞细胞增强抗原呈递，进一步协同增强机体的免疫反应，展现出更强的抗肿瘤活性。
总之，本发明提供了一种花菁类染料介导的核酸/阿霉素复合药物的制备方法，其首次将花菁类荧光分子引入核酸的自组装过程，方法简单，所制备的药物负载率高。同时，所制备的复合药物具有良好的血液循环稳定性和弱酸响应性，在体内肿瘤区域过表达GSH和H2O2的条件下易降解，可以缓慢释放DOX和CPG。更重要的是，DOX可以诱导细胞免疫原性死亡，结合免疫佐剂CPG唤醒机体的免疫反应，展现出更高的抗肿瘤活性。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
Claims (6)
1.一种花菁染料介导的核酸/抗癌药物复合物的制备方法，其特征在于：将花菁染料修饰的功能核酸和抗癌化疗药物在水溶液中自组装成尺寸均匀、负载率高的新型功能核酸/抗癌药物复合物；其具体包括以下步骤：
（1）花菁染料修饰的功能核酸原液的制备：将花菁染料修饰的功能核酸溶解在无菌水中，室温、避光条件下吹打均匀，形成均匀的花菁染料修饰的功能核酸原液；
（2）药物原液的制备：将抗癌化疗药物溶解在溶剂中，室温、避光条件下混合均匀，得到药物原液；
（3）核酸/抗癌药物复合物的制备：将花菁染料修饰的功能核酸原液与药物原液混合均匀，置于金属浴中，95 ℃反应1 h，然后冷却至室温，再将产物离心，二次水洗两次，制备得到所述核酸/抗癌药物复合物；
所述抗癌化疗药物为盐酸佐柔比星、盐酸阿霉素或阿霉素的结构类似物及衍生物。
2. 根据权利要求1所述的一种花菁染料介导的核酸/抗癌药物复合物的制备方法，其特征在于：步骤（1）所得花菁染料修饰的功能核酸原液的浓度为100 μM。
3.根据权利要求1所述的一种花菁染料介导的核酸/抗癌药物复合物的制备方法，其特征在于：步骤（2）中所述溶剂包括水、二甲基亚砜中的任意一种。
4. 根据权利要求1所述的一种花菁染料介导的核酸/抗癌药物复合物的制备方法，其特征在于：步骤（2）所得药物原液的浓度为10 mM。
5.根据权利要求1所述的一种花菁染料介导的核酸/抗癌药物复合物的制备方法，其特征在于：所用花菁染料修饰的功能核酸与抗癌化疗药物的摩尔比为1:30。
6. 根据权利要求1所述的一种花菁染料介导的核酸/抗癌药物复合物的制备方法，其特征在于：步骤（3）中离心的转速为8000 rpm，时间为5 min。
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