CN110251531B - 一种用于治疗肿瘤的靶向系统及其制备方法与应用 - Google Patents
一种用于治疗肿瘤的靶向系统及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供一种用于治疗肿瘤的靶向系统及其制备方法与应用。靶向系统含有Apt‑CS/rGO/Ag+‑DNA复合纳米材料，其中，Apt‑CS表示对所述肿瘤有特异性的核酸适配体‑壳聚糖耦合物；Ag+‑DNA表示Ag+‑DNA耦合物；rGO表示还原型氧化石墨烯。靶向系统1)在制备真核生物肿瘤细胞增殖抑制剂中的应用；2)在制备治疗肿瘤药物中的应用也属于本发明的保护范围。本发明制备了特异性核酸适配体负载Ag+/DNA/rGO复合纳米材料，将Ag+靶向运输到肿瘤部位；在肿瘤细胞中，在维生素C刺激下，rGO协同H2O2共同催化Ag+还原成Ag纳米颗粒，利用Ag纳米颗粒的生物学毒性，从而实现对肿瘤的治疗作用。
Description
技术领域
本发明属于医药领域，具体涉及一种用于治疗肿瘤的靶向系统及其制备方法与应用。
背景技术
如今，肿瘤已经成为了威胁中国居民生命健康最大的因素，无论男性还是女性，恶性肿瘤死亡率均逐渐升高。目前，肿瘤治疗效果差、复发转移率高且肿瘤治疗副作用大、精准性差等原因，导致肿瘤治疗难度大。因此，聚焦我国高发、特色和具有优势研究基础的癌种，并针对其进行相应的靶向治疗是提高患者生存率的一个关键突破点。
银纳米颗粒可以作为抗菌剂和抗癌剂，并被广泛应用于生物医学领域。它可以选择性地破坏胞内线粒体呼吸链，引起活性氧簇ROS的积累和中断ATP的生成，来造成核酸链损伤。
利用银纳米颗粒来治疗肿瘤迫切需要解决的问题是如何将银纳米颗粒靶向引导到肿瘤部位。
发明内容
本发明的目的是提供一种用于治疗肿瘤的靶向系统及其制备方法。
本发明所提供的用于治疗肿瘤的靶向系统，含有Apt-CS/rGO/Ag+-DNA复合纳米材料，其中，Apt-CS表示对所述肿瘤有特异性的核酸适配体-壳聚糖耦合物；Ag+-DNA表示Ag+-DNA耦合物；rGO表示还原型氧化石墨烯；
所述肿瘤为癌，所述癌为人乳腺癌时；所述核酸适配体为核仁素适配体AS1411，具体可为Cy5荧光标记的核仁素适配体AS1411。
所述癌为人肺癌时；所述核酸适配体为Endoglin适配体或核仁素适配体AS1411，具体可为Cy5荧光标记的Endoglin适配体或核仁素适配体AS1411。
所述癌为肝癌时，所述核酸适配体为TLS11a，具体可为Cy5荧光标记的TLS11a适配体。
所述DNA用于负载银离子，并与石墨烯通过π-π共轭形成复合纳米材料；
所述DNA的序列为：GCCGCGTGCGGCCGGTGCCGAGAGAGAGAGAGGAGAGAGA。
所述Apt-CS/rGO/Ag+-DNA复合纳米材料的厚度可为30-40nm。
所述Apt-CS/rGO/Ag+-DNA复合纳米材料通过包括如下步骤的方法制备得到：
1)制备Ag+-DNA耦合物溶液；
2)制备还原型氧化石墨烯溶液；
3)将还原型氧化石墨烯溶液加入到Ag+-DNA偶合物溶液中，涡旋，孵育，离心，收集沉淀得到rGO/Ag+-DNA，将rGO/Ag+-DNA分散到水中得到rGO/Ag+-DNA溶液；
4)将壳聚糖分散到冰醋酸溶液中，加入rGO/Ag+-DNA溶液，搅拌反应，离心，收集沉淀，得到CS/rGO/Ag+-DNA纳米材料，将核酸适配体与CS/rGO/Ag+-DNA纳米材料中的CS偶联，得到Apt-CS/rGO/Ag+-DNA复合纳米材料。
上述方法步骤1)中，制备Ag+-DNA耦合物溶液的操作如下：将硝酸银溶解于水中，将DNA加入到硝酸银溶液中，涡旋，孵育，得到Ag+-DNA耦合物溶液；
其中，硝酸银中银离子与DNA的配比可为：0.2mmol：1OD(体积10μL)；
所述涡旋的时间可为5-10min；
所述孵育的温度可为22-27℃，时间可为1-2h；
上述方法步骤2)中，制备还原型氧化石墨烯溶液的操作包括：将氧化石墨分散到水中，超声剥离得到氧化石墨烯片，采用水合肼还原，得到还原型氧化石墨烯(rGO)溶液。
上述方法步骤3)中，所述还原型氧化石墨烯的溶液中还原型氧化石墨烯与Ag+-DNA偶合物溶液的配比可为：0.05mg：1mL；其中，1mL Ag+-DNA偶合物溶液为通过将0.2mmol的硝酸银溶解于1mL水中，将1OD DNA加入到所述硝酸银溶液孵育后制得的溶液；
所述涡旋的时间可为5-10min；
所述孵育的温度可为22-27℃，时间可为30-40min；
所述离心的条件为：10000res/min离心10min；
上述方法步骤4)中，所述冰醋酸溶液可为质量浓度1％的冰醋酸溶液；
所述壳聚糖与rGO/Ag+-DNA溶液的配比可为：1mg：1mL；其中，1mL rGO/Ag+-DNA溶液通过将50μL 1mg/mL还原型氧化石墨烯溶液加入到1mL Ag+-DNA偶合物溶液中孵育，去除上清液，再将得到的GO/Ag+-DNA沉淀分散到1mL水中制得；
所述搅拌反应的时间可为6h；
所述离心的条件可为：12000res/min 15min；
步骤4)中，所述偶联以戊二醛为偶联剂实现；
所述戊二醛以溶液的形式加入，所述戊二醛溶液的质量浓度可为2.5％；
所述核酸适配体的浓度可为1OD；
所述CS/rGO/Ag+-DNA纳米材料中的CS与核酸适配体的配比可为：1mg：100μL。
上述靶向系统1)在制备真核生物肿瘤细胞增殖抑制剂中的应用；2)在制备治疗肿瘤药物中的应用也属于本发明的保护范围。
所述真核生物为哺乳动物；
所述肿瘤细胞为癌细胞；
所述癌细胞可为乳腺癌细胞或肺癌细胞；
所述乳腺癌细胞具体可为人乳腺癌细胞MCF-7；
所述肺癌细胞具体可为人肺癌细胞A549。
所述肿瘤为癌；所述癌可为乳腺癌或肺癌。
含有上述靶向系统，即CS/rGO/Ag+-DNA纳米材料的真核生物肿瘤细胞增殖抑制剂或治疗肿瘤的药物也属于本发明的保护范围。
所述真核生物肿瘤细胞增殖抑制剂或治疗肿瘤的药物可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织；或是被其他物质混合或包裹后导入机体。
需要的时候，在上述药物中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
以上述靶向系统，即CS/rGO/Ag+-DNA纳米材料为活性成分制备的真核生物肿瘤细胞增殖抑制剂或治疗肿瘤药物可以制成注射液、片剂、粉剂、颗粒剂、胶囊、口服液、膏剂、霜剂等多种形式。上述各种剂型的药物均可以按照药学领域的常规方法制备。
本发明制备了特异性核酸适配体负载Ag+/DNA/rGO复合纳米材料，将Ag+靶向运输到肿瘤部位；在肿瘤细胞中，在维生素C(Vc)刺激下，rGO协同H2O2共同催化Ag+还原成Ag纳米颗粒，利用Ag纳米颗粒的生物学毒性，从而实现对肿瘤的治疗作用。
附图说明
图1为本发明实施例制备的Apt-CS/rGO/Ag+-DNA复合纳米材料的XPS表征图
图2为本发明实施例制备的Apt-CS/rGO/Ag+-DNA复合纳米材料原子力显微镜表征图。
图3为100μL 1mg/mL Apt-CS/rGO/Ag+-DNA复合纳米材料与MCF-7细胞孵育后，PBS洗涤，加入100μL 1mg/mL PI染料，在10μL 5mM Vc刺激下随着时间推移，细胞释放过氧化氢催化生成银单质的共聚焦显微镜图。
图4为PBS(0.01M，pH＝7.4)100μL、Vc(0.1M)100μL、RS--CS/rGO/Ag+-DNA(1mg/mL)100μL、RS-CS/rGO/Ag+-DNA(1mg/mL)100μL及Vc(0.1M)100μL、Apt-CS/rGO/Ag+-DNA(1mg/mL)100μL、Apt-CS/rGO/Ag+-DNA(1mg/mL)100μL及Vc(0.1M)100μL复合材料治疗荷瘤鼠的肿瘤体积生长曲线。
图5为100μL PBS、100μL 1mg/mL RS-CS/rGO/Ag+-DNA复合纳米材料、100μL 1mg/mL Apt-CS/rGO/Ag+-DNA复合纳米材料不同时间的体内肿瘤靶向荧光活体成像图。
具体实施方式
下面通过具体实施例对本发明进行说明，但本发明并不局限于此。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、生物材料等，如无特殊说明，均可从商业途径得到。
下述实施例中采用的DNA为上海生工公司的产品，其序列为：GCCGCGTGCGGCCGGTGCCGAGAGAGAGAGAGGAGAGAGA
实施例1、Apt-CS/rGO/Ag+-DNA复合纳米材料的制备
1)称取34mg硝酸银溶解于1mL的超纯水，将溶解的10μL 1OD DNA加入硝酸银溶液，涡旋5min，室温孵育1h；
2)取1mg氧化石墨烯(GO)溶于1mL超纯水，利用细胞破碎超声仪超声6h，3000res/min离心，去除沉淀，加入10μL水合肼(NH2-NH2)，涡旋10min，60℃水浴4h，离心10000rpm，去除上清液，洗涤两次，分散成1mg/mL还原型氧化石墨烯溶液备用；
3)取50μL还原性石墨烯溶液加入1mL步骤1)制备的Ag+-DNA溶液中，涡旋5min，室温孵育30min，10000res/min离心10min，去除上清液，洗涤两次，合成GO/Ag+-DNA，分散于1mL超纯水中备用；
4)取10mg壳聚糖(CS)分散于10mL质量浓度1％的冰醋酸溶液中，取1mL加入10mL超纯水，搅拌溶解，直到溶液无气泡产生，然后加入1mL上述rGO/Ag+-DNA溶液，搅拌反应6h，离心12000res/min 15min去除上清液，得到CS/rGO/Ag+-DNA纳米材料，加入50μL质量浓度2.5％戊二醛溶液和100μL 1OD Cy5荧光标记的核仁素适配体AS1411，搅拌反应1h，加入1％的BSA搅拌30min，12000res/min离心去除上清液，洗涤2次，得到Apt-CS/rGO/Ag+-DNA复合纳米材料。
图1为XPS表征图，a为Apt-CS/rGO/Ag+-DNA复合纳米材料，b为rGO纳米材料。
利用XPS表征分析检测rGO、Apt-CS/rGO/Ag+-DNA复合纳米材料的元素组成。从图1可以看出，Apt-CS/rGO/Ag+-DNA复合纳米材料组成元素中含有P、Ag、C、O、N等，而rGO中有的C、O、N元素，表明该复合纳米材料合成成功。
图2为Apt-CS/rGO/Ag+-DNA复合纳米材料原子力显微镜表征图。
利用AFM显微镜观察Apt-CS/rGO/Ag+-DNA复合纳米材料。从图2可以看出，Apt-CS/rGO/Ag+-DNA复合纳米材料厚度大约为30-40nm。
2、复合纳米材料的活性研究
复合纳米材料与细胞靶向结合：
将细胞离心重悬均匀的铺于孔板中，培养6h，计数，用吸管将培养基去除，PBS洗涤两次，加入100μL Apt-CS/rGO/Ag+-DNA复合纳米材料，轻微晃动孔板，使纳米材料均匀分布于细胞，孵育10min，PBS洗涤两次，加入10μL 5mM Vc，轻微晃动孔板，使Vc溶液均匀分布于细胞，显微镜观察细胞靶向性及银沉积现象。
PI(碘化丙啶)染料观测细胞死亡：
将MCF-7、293T细胞离心重悬均匀的铺于孔板中，培养6h，计数，用吸管将培养基去除，PBS洗涤两次，加入终浓度2mg/mL Apt-CS/rGO/Ag+-DNA复合纳米材料，轻微晃动孔板，使纳米材料均匀分布于细胞，孵育10min，PBS洗涤两次，加入终浓度为5μg/mL PI染料，轻微晃动孔板，加入终浓度为2mMVc，轻微晃动孔板，使Vc溶液均匀分布于细胞，共聚焦显微镜观察细胞形态。
图3为终浓度2mg/mL Apt-CS/rGO/Ag+-DNA复合纳米材料与MCF-7(A)或293T(B)细胞孵育后，PBS洗涤，加入终浓度5μg/mL PI染料，在终浓度2mM Vc刺激下随着时间推移，细胞释放过氧化氢催化生成银单质的共聚焦显微镜图。
体内抗肿瘤研究：
荷瘤鼠造模：选择4-6周龄的Balb/c Nude鼠，收集对数生长期的A549人肺癌细胞，PBS重悬将细胞浓度调至1×107个/mL；取200μL细胞悬液皮下注射小鼠右肢上侧，观察1-2周。
肿瘤治疗：收集A549细胞，将约2×107的细胞注射到裸鼠背部皮下；采取随机数字表的方法，将荷瘤鼠随机分为4组，每组6只；待肿瘤体积长至约为100mm3时(种瘤10天后)，每隔3天尾静脉注射纳米材料及Vc，计量如下：PBS(0.01M，pH＝7.4)100μL、Vc(0.1M)100μL、RS--CS/rGO/Ag+-DNA(RS是指随机DNA链，其序列为ATACCAGCTTATTCAATTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGATAGTAAGTGCAATCT：主要是作对照实验用的，也是由上海生工公司提供)(1mg/mL)100μL、RS-CS/rGO/Ag+-DNA(1mg/mL)100μL及Vc(0.1M)100μL、Apt-CS/rGO/Ag+-DNA(1mg/mL)100μL、Apt-CS/rGO/Ag+-DNA(1mg/mL)100μL及Vc(0.1M)100μL，计算肿瘤体积，绘制肿瘤体积生长曲线。
体内成像：收集A549细胞，将约1×107的细胞注射到裸鼠背部皮下；采取随机数字表的方法，将荷瘤鼠随机分为4组，每组8只；待肿瘤体积长至约为100mm3时(种瘤10天后)，分别尾静脉注射纳米材料及Vc，计量如下：PBS(0.01M，pH＝7.4)100μL、Apt-CS/rGO/Ag+-DNA(1mg/mL)100μL、RS-CS/rGO/Ag+-DNA(1mg/mL)100μL，2h后尾静脉注射Vc(10mM)100μL，观察复合纳米材料在小鼠体内的荧光动态分布。
图4为PBS(0.01M，pH＝7.4)100μL、Vc(0.1M)100μL、RS--CS/rGO/Ag+-DNA(1mg/mL)100μL、RS-CS/rGO/Ag+-DNA(1mg/mL)100μL及Vc(0.1M)100μL、Apt-CS/rGO/Ag+-DNA(1mg/mL)100μL、Apt-CS/rGO/Ag+-DNA(1mg/mL)100μL及Vc(0.1M)100μL复合材料治疗荷瘤鼠的肿瘤体积生长曲线。
图5为100μL PBS、100μL 1mg/mL RS-CS/rGO/Ag+-DNA复合纳米材料、100μL 1mg/mL Apt-CS/rGO/Ag+-DNA复合纳米材料不同时间的体内肿瘤靶向荧光活体成像图。
Claims (11)
1.一种用于治疗肿瘤的靶向制剂，含有：Apt-CS/rGO/Ag+-DNA复合纳米材料，其中，Apt-CS表示对所述肿瘤有特异性的核酸适配体-壳聚糖耦合物；Ag+-DNA表示Ag+-DNA耦合物；rGO表示还原型氧化石墨烯。
2.根据权利要求1所述的靶向制剂，其特征在于：所述肿瘤为癌，所述癌为人乳腺癌，所述核酸适配体为核仁素适配体AS1411；
或所述癌为人肺癌，所述核酸适配体为核仁素适配体AS1411或Endoglin适配体；
或所述癌为肝癌，所述核酸适配体为TLS11a。
3.根据权利要求1或2所述的靶向制剂，其特征在于：所述Apt-CS/rGO/Ag+-DNA复合纳米材料的厚度为30-40nm。
4.制备权利要求1-3中任一项所述的靶向制剂的方法，包括如下步骤：
1)制备Ag+-DNA耦合物溶液；
2)制备还原型氧化石墨烯溶液；
3)将还原型氧化石墨烯溶液加入到Ag+-DNA耦合物溶液中，涡旋，孵育，离心，收集沉淀得到rGO/Ag+-DNA，将rGO/Ag+-DNA分散到水中得到rGO/Ag+-DNA溶液；
4)将壳聚糖分散到冰醋酸溶液中，加入rGO/Ag+-DNA溶液，搅拌反应，离心，收集沉淀，得到CS/rGO/Ag+-DNA纳米材料，将适配体与CS/rGO/Ag+-DNA纳米材料中的CS偶联，得到Apt-CS/rGO/Ag+-DNA复合纳米材料。
5.根据权利要求4所述的方法，其特征在于：步骤1)中，制备Ag+-DNA耦合物的操作如下：将硝酸银溶解于水中，将DNA加入到硝酸银溶液中，涡旋，孵育，得到Ag+-DNA耦合物溶液；
其中，硝酸银中银离子与DNA的配比为：0.2mmol：1OD；
所述涡旋的时间为5-10min；
所述孵育的温度为22-27℃，时间为1-2h。
6.根据权利要求4或5所述的方法，其特征在于：步骤3)中，所述还原型氧化石墨烯的溶液中还原型氧化石墨烯与Ag+-DNA偶合物溶液的配比为：0.05mg：1mL；
其中，1mL Ag+-DNA耦合物溶液为通过将0.2mmol的硝酸银溶解于1mL水中，将1OD DNA加入到所述硝酸银溶液孵育后制得的溶液；
所述涡旋的时间为5-10min；
所述孵育的温度为22-27℃，时间为30-40min；
所述离心的条件为：10000res/min离心10min。
7.根据权利要求4或5所述的方法，其特征在于：步骤4)中，所述冰醋酸溶液为质量浓度1％的冰醋酸溶液；
所述壳聚糖与rGO/Ag+-DNA溶液的配比为：1mg：1mL；
其中，1mL rGO/Ag+-DNA溶液通过将50μL 1mg/mL还原型氧化石墨烯溶液加入到1mL Ag+-DNA耦合物溶液中孵育，去除上清液，再将得到的rGO/Ag+-DNA沉淀分散到1mL水中制得；
所述搅拌反应的时间为6h；
所述离心的条件为：12000res/min 15min。
8.根据权利要求4或5所述的方法，其特征在于：步骤4)中，所述偶联以戊二醛为偶联剂实现；
所述戊二醛以溶液的形式加入，所述戊二醛溶液的质量浓度为2.5％；
所述核酸适配体的浓度为1OD；
所述CS/rGO/Ag+-DNA纳米材料中的CS与核酸适配体的配比为：1mg：100μL。
9.权利要求1-3中任一项所述的靶向制剂1)在制备真核生物肿瘤细胞增殖抑制剂中的应用；所述真核生物为哺乳动物；所述肿瘤细胞为癌细胞；
或2)在制备治疗肿瘤药物中的应用；
所述肿瘤为癌。
10.根据权利要求9所述的应用，其特征在于：所述癌细胞为乳腺癌细胞、肺癌细胞或肝癌细胞；
所述癌为乳腺癌、肺癌或肝癌。
11.含有权利要求1-3中任一项所述的靶向制剂的真核生物肿瘤细胞增殖抑制剂或治疗肿瘤的药物。
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