CN113384694A - 一种用于肿瘤诊疗一体化的纳米复合药物及其制备方法 - Google Patents
一种用于肿瘤诊疗一体化的纳米复合药物及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于肿瘤诊疗一体化的纳米复合药物及其制备方法，该药物包括双面神结构探针、化疗药物和光动力药物，所述双面神结构探针由ATP适体DNA、金纳米粒子，以及具有靶向和载药功能的DNA通过点击反应合成的一面为靶向和载药功能DNA，一面为ATP适体DNA的双面神结构的探针，化疗药物和光动力药物加载在具有靶向和载药功能DNA上。其利用在纳米间隙中发生的显著增强的等离子体耦合导致的电磁热点，显著提高拉曼信号，在单个活细胞内实现了ATP的检测与成像分析，且采用肿瘤细胞成像的方式，可视化监测肿瘤细胞，实现诊疗一体化。
Description
技术领域
本发明属于肿瘤细胞诊疗一体技术领域，尤其涉及一种用于肿瘤诊疗一体化的纳米复合药物及其制备方法。
背景技术
目前，最接近的现有技术：
现有技术一：(Zhou W,Li Q,Liu H,et al.Building Electromagnetic HotSpots in Living Cells via Target-Triggered Nanoparticle Dimerization[J].AcsNano,2017,11(4):3532.)中提出：设计了一种目标介导的纳米二聚化策略，以在活细胞中建立电磁热点，从而能够实时进行细胞内微小RNA(miRNA)的无背景多重成像。目标miRNA诱导结构上可重现的二聚体纳米结构的形成，其中新产生的电磁热点可显着增强拉曼散射，并且应用于对肿瘤细胞和正常细胞的检测。然而，该方法并未涉及治疗方面。
现有技术二：(Hyeongmok Park,Jinhwan Kim,Sungjin Jung,et al.DNA-AuNanomachine Equipped with i-Motif and G-Quadruplex for Triple CombinatorialAnti-Tumor Therapy.Adv.Funct.Mater,2018,28,1705416)中提到设计了一种功能性DNA装饰的动态金(Au)纳米机器来进行三联组合抗癌治疗。利用Au纳米颗粒的固有光学特性(取决于其大小)，将富含胞嘧啶的i-基序序列用于细胞内pH敏感的双链解离和随后的DNA-Au纳米机器聚集，从而实现在用红外光照射时抗癌药物释放和光热消融。此外，另一种功能性DNA序列G-四链体被用于稳定加载和递送光敏剂，从而在红光照射下实现有效的光动力疗法。研究了pH响应的动态聚集行为，随后的药物释放以及光热效应并且评估了治疗效果。然而，该方法并未涉及细胞中生物标志物的检测，而且在细胞治疗方面并没有靶向治疗的作用。
综上所述，现有技术存在的问题是：
(1)现有技术的目标介导的纳米二聚化探针只是进行荧光成像和拉曼成像监测，没有制备双功能的双面神纳米粒子，不能进行诊疗一体化研究。
(2)现有技术是利用肿瘤细胞的强胞吞胞吐作用进行探针的富集，然后进入细胞内进行检测和治疗，缺少靶向性。
解决上述技术问题的难度：
(1)利用“点击反应”合成多功能纳米粒子，且具有较好的生物相容性，同时发挥肿瘤的诊断与治疗作用。
(2)活细胞的快速成像与检测。细胞脱离培养箱环境，存活概率会大大降低，构建的探针需要低毒性，且能与细胞快速、特异性的作用。
现有技术中目标介导的纳米二聚化的探针没有被应用于肿瘤细胞检测与靶向治疗相结合，检测方法单一，另外现有技术是利用肿瘤细胞的强胞吞胞吐作用进行探针的富集，然后进入细胞内进行检测和治疗，缺少靶向性。因此需要制备一种新型探针来解决上述技术问题。
发明内容
针对现有技术存在的问题，本发明提供了一种用于肿瘤诊疗一体化的纳米复合药物及其制备方法。
为了实现上述目的，本发明采用以下技术方案：
一种用于肿瘤诊疗一体化的纳米复合药物，包括双面神结构探针、化疗药物和光动力药物，所述双面神结构探针包括依次连接的巯基ATP适体DNA、AuNP、SH-PEG-DBCO和具有靶向和载药功能的DNA双链，化疗药物和光动力药物直接负载在具有靶向和载药功能DNA双链上，具有靶向和载药功能的DNA双链由带叠氮基修饰具有靶向功能DNA及其互补链构成。
具体地，所述带叠氮基修饰具有靶向功能DNA序列为(N3)-GGC GGA GCC CTA ACCCTA ACC CTA ACC CTG GTG GTG GTG GTT GTG GTG GTG GTG G-3’；带叠氮基修饰具有靶向功能DNA互补链序列为TAGGGCTCCGCC-3’；ATP适体DNA由(SH)-TTTTTTTTTACCTGGGGGAGTAT和(SH)-TTTTTTTTTTGCGGAGGAAGGT组成。
一种用于肿瘤诊疗一体化的纳米复合药物的制备方法，具体包括以下步骤：
第一步，制备成一面具有靶向和载药功能DNA，一面ATP适体DNA的双面神结构的探针；
第二步，加载化疗药物和光动力药物。
具体地，双面神结构探针制备方法为：
(1)采用现有技术合成50nm的金纳米粒子，通过将氨基涂覆的载玻片浸入AuNPs溶液中过夜，将AuNPs吸附到载玻片上；然后将载玻片浸入一端修饰巯基，一端修饰环炔的巯基聚乙二醇二苯并环辛炔(SH-PEG-DBCO)中一段时间，超声处理后分散在磷酸盐缓冲液中；
(2)将带叠氮基修饰具有靶向功能DNA与其互补链按1：1.2混合并在95℃下退火5分钟，形成具有靶向和载药功能的DNA双链；
(3)通过点击反应与超声处理使具有靶向和载药功能的DNA双链与一面修饰SH-PEG-DBCO的AuNPs相连；
(4)再利用Au-S键将TCEP处理后的巯基ATP适体DNA连接在AuNP上，在12小时内向所得溶液中缓慢添加NaCl进行老化，离心去除过量的DNA，得到一面具有靶向和载药功能DNA，一面ATP适体DNA的双面神结构探针。
所述化疗药物包括但不限于DOX、柔红霉素和表柔比星中的一种或多种，所述光动力药物包括但不限于TMPyP4、酞箐锌和酞箐铜中的一种或多种。
进一步，所述加载化疗药物DOX和光动力药物TMPyP4的方法包括：将化疗药物DOX与制备的双面神结构探针Au-I混合三小时以上，反应结束后通过在8000r离心5分钟从溶液中去除过量的药物并分散在Tris buffer(三羟甲基氨基甲烷缓冲液)中，得到Au-I-DOX，将TMPyP4与Au-I-DOX探针混合通过在7500rpm下离心5分钟除去游离的TMPyP4，得到Au-I-D/T。
与现有技术相比，本发明具有以下有益效果：(1)构建了一种肿瘤细胞中高含量ATP的检测方法以及对肿瘤细胞的协同治疗方法，利用在纳米间隙中发生的显著增强的等离子体耦合导致的电磁热点，显著提高拉曼信号，在单个活细胞内实现了ATP的检测与成像分析，达到了对肿瘤标志物、活细胞等物质在生命分析方面的灵敏检测的要求；(2)荧光光谱法具有灵敏度高、线性范围大、检测所需样品少等特点，荧光成像分析可以直观观测活细胞等生物样品，本发明利用荧光进行治疗效果评估，采用肿瘤细胞成像的方式，可视化监测肿瘤细胞，实现诊疗一体化；(3)通过目标介导的纳米二聚化技术与靶向治疗技术提高了对肿瘤细胞及其标志物靶向检测的准确性和灵敏度，能够达到早期诊断治疗的目的，这对肿瘤诊断、监测和治疗方面具有重要意义。
附图说明
图1是实施例1涉及的用于肿瘤诊疗一体化的纳米复合药物的制备方法的原理图。
图2是双面神结构探针与ATP进行体外拉曼检测结果图，其中A为单个AuNPsTEM图，B为AuNPs二聚体的TEM图，C为单个AuNPs(a)和AuNPs二聚体(b)的紫外-可见光谱图，D为DTNB在单个AuNP(a)和AuNPs二聚体(b)上的SERS信号图。
图3是双面神结构探针与ATP进行体外拉曼检测的SERS成像图，从左到右依次为细胞的明场图，荧光图，叠加图，从上向下依次对应不同孵育时间(2，4，6，8h)、单个AuNPs粒子和AuNPs二聚体。
图4是图4单个AuNPs粒子(左)和AuNPs二聚体(右)SERS成像图中a、b、c和d所指位置的拉曼信号对比图。
图5为多种探针进入肿瘤细胞内，对肿瘤细胞进行的ROS荧光成像图。
图6为不同探针进入肿瘤细胞内进行细胞毒性实验结果图。
图7为不同探针进入肿瘤细胞内进行细胞毒性实验结果图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例1：
本实施例涉及的一种用于肿瘤诊疗一体化的纳米复合药物，包括双面神结构探针、化疗药物和光动力药物，所述双面神结构探针包括依次连接的巯基ATP适体DNA、AuNP、SH-PEG-DBCO和具有靶向和载药功能的DNA双链，化疗药物和光动力药物直接负载在具有靶向和载药功能DNA双链上，具有靶向和载药功能的DNA双链由带叠氮基修饰具有靶向功能DNA及其互补链构成。
具体地，所述带叠氮基修饰具有靶向功能DNA序列为(N3)-GGC GGA GCC CTA ACCCTA ACC CTA ACC CTG GTG GTG GTG GTT GTG GTG GTG GTG G-3’；带叠氮基修饰具有靶向功能DNA互补链序列为TAGGGCTCCGCC-3’；ATP适体DNA由(SH)-TTTTTTTTTACCTGGGGGAGTAT和(SH)-TTTTTTTTTTGCGGAGGAAGGT组成。其他带叠氮基修饰具有靶向功能DNA和ATP适体DNA也可以为其他能够实现上述功能的结构。
本实施例涉及的一种用于肿瘤诊疗一体化的纳米复合药物的制备方法，具体包括以下步骤：
1、合成双面神结构探针(Au-I)：
(1)采用现有技术合成50nm的金纳米粒子，通过将氨基涂覆的载玻片浸入AuNPs溶液中过夜，将AuNPs吸附到载玻片上；然后将载玻片浸入一端修饰巯基，一端修饰环炔的巯基聚乙二醇二苯并环辛炔(SH-PEG-DBCO)中2小时，再超声处理2分钟，超声后分散在磷酸盐缓冲液(pH7.4，吐温0.01％)中；
(2)将带叠氮基修饰具有靶向功能DNA与其互补链按1：1.2混合并在95℃下退火5分钟，形成具有靶向和载药功能的DNA双链；
(3)通过点击反应(Click)与超声处理(ultrasound)使具有靶向和载药功能的DNA双链与一面修饰SH-PEG-DBCO的AuNPs相连；
(4)再利用Au-S键将TCEP处理后的巯基ATP适体DNA连接在AuNP上，在12小时内向所得溶液中缓慢添加NaCl进行老化，8000r离心5分钟去除过量的DNA，得到一面具有靶向和载药功能DNA，一面ATP适体DNA的双面神结构探针，将其分散在三乙酸盐缓冲液(pH 7.4)中保存待用。
2、加载药物
(1)加载化疗药物DOX制备Au-I-DOX
将化疗药物DOX与制备的双面神结构探针Au-I混合三小时以上，反应结束后通过在8000r离心5分钟从溶液中去除过量的药物并分散在Tris buffer(三羟甲基氨基甲烷缓冲液)中，得到Au-I-DOX。
(2)加载光动力药物TMPyP4制备Au-I-TMPyP4
将TMPyP4与双面神结构探针Au-I混合，在7500rpm下离心5分钟除去游离的TMPyP4，得到Au-I-TMPyP4。
(3)加载化疗药物DOX和光动力药物TMPyP4制备Au-I-D/T
将TMPyP4与Au-I-DOX探针混合通过在7500rpm下离心5分钟除去游离的TMPyP4，得到Au-I-D/T。
并使用特定的单线态氧猝灭剂1,3-二苯基异苯并呋喃(DPBF)评估TMPyP4的ROS生成。
3.双面神结构的探针与ATP反应，进行体外拉曼检测
将探针分子5,5'-二硫代双-(2-硝基苯甲酸)(DTNB)与制备的双面神结构探针Au-I一起孵育过夜，然后加入ATP反应2小时形成AuNPs二聚体(Dimers)。反应结束后取1μL样品干燥后在金片上进行表面增强拉曼SERS检测。
拉曼检测数据的处理：拉曼探针分子的拉曼特征吸收峰为1320cm-1，分别对其出峰位置的出峰强度进行扣除空白处理进行分析，结果如图2所示。从图2B中可以看出AuNPs具有明显的二聚行为，从2C也可以看出，与单个AuNPs(a)相比，AuNPs二聚体(b)在530nm附近吸收峰减弱，在680nm左右出现新峰。这是因为AuNPs二聚体的纳米间隙中发生了显著增强的等离子体耦合，从而导致了电磁热点的形成，即可以显着提高拉曼信号。从图2D可以明显地看出，AuNPs二聚体(b)的拉曼信号比单个金胶的拉曼信号强7倍以上。
4、细胞内拉曼成像
(1)肿瘤细胞的培养：使用RPMI 1640培养液在37℃培养箱(5％的二氧化碳和95％的空气)中对MCF-7细胞进行培养，每天更换培养液。癌细胞的生长培养过程要注意杀菌消毒，时刻关注癌细胞的生长状态。
(2)探针与肿瘤细胞孵育，进行细胞的拉曼成像：
将消化后的肿瘤细胞接种在金片上，加入50μL双面神结构探针Au-I进行不同时间(2，4，6，8h)的孵育；将细胞培养皿固定到显微镜载物台上，在细胞培养的状态下使用拉曼光谱仪的633nm激光器，并用50倍物镜进行SERS细胞成像，结果如图3和4所示。
(3)探针与肿瘤细胞孵育，进行ROS的荧光成像、细胞毒性和流式细胞实验
将细胞消化后接种到小皿里，待细胞在小皿中长至均匀后，分别与Au-I探针(Au-I)、化疗药物DOX、光动力药物TMPyP4、加载DOX的Au-I探针(Au-I-DOX)、加载TMPyP4的Au-I探针(Au-I-TMPyP4)和同时加载DOX和TMPyP4的Au-I探针(Au-I-D/I)反应6h，然后将细胞用PBS溶液清洗三次防止多余的探针增加荧光背景信号。
采用共聚焦显微镜对上述处理后的部分细胞进行ROS的荧光成像测试，结果如图5所示，结果表明加载光敏剂(TMPyP4)的探针产生的ROS最多。
对上述处理后的部分细胞进行细胞毒性实验，结果如图6所示。对上述处理后的部分细胞进行流式细胞实验，结果如图7所示。结果表明，同时加载两种药物的探针治疗效果最好。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
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Claims (4)
1.一种用于肿瘤诊疗一体化的纳米复合药物，其特征在于，包括双面神结构探针、化疗药物和光动力药物，所述双面神结构探针包括依次连接的巯基ATP适体DNA、AuNP、SH-PEG-DBCO和具有靶向和载药功能的DNA双链，化疗药物和光动力药物直接负载在具有靶向和载药功能DNA双链上，具有靶向和载药功能的DNA双链由带叠氮基修饰具有靶向功能DNA及其互补链构成。
2.根据权利要求1所述的用于肿瘤诊疗一体化的纳米复合药物，其特征在于，所述带叠氮基修饰具有靶向功能DNA序列为(N3)-GGC GGA GCC CTA ACC CTA ACC CTA ACC CTG GTGGTG GTG GTT GTG GTG GTG GTG G-3’；带叠氮基修饰具有靶向功能DNA互补链序列为TAGGGCTCCGCC-3’；ATP适体DNA由(SH)-TTTTTTTTTACCTGGGGGAGTAT和(SH)-TTTTTTTTTTGCGGAGGAAGGT组成。
3.一种用于肿瘤诊疗一体化的纳米复合药物的制备方法，其特征在于，具体包括以下步骤：
(1)采用现有技术合成50nm的金纳米粒子，通过将氨基涂覆的载玻片浸入AuNPs溶液中过夜，将AuNPs吸附到载玻片上；然后将载玻片浸入一端修饰巯基，一端修饰环炔的巯基聚乙二醇二苯并环辛炔中一段时间，超声处理后分散在磷酸盐缓冲液中；
(2)将带叠氮基修饰具有靶向功能DNA与其互补链按1：1.2混合并在95℃下退火5分钟，形成具有靶向和载药功能的DNA双链；
(3)通过点击反应与超声处理使具有靶向和载药功能的DNA双链与一面修饰SH-PEG-DBCO的AuNPs相连；
(4)再利用Au-S键将TCEP处理后的巯基ATP适体DNA连接在AuNP上，在12小时内向所得溶液中缓慢添加NaCl进行老化，离心去除过量的DNA，得到一面具有靶向和载药功能DNA，一面ATP适体DNA的双面神结构探针。
(5)加载化疗药物和光动力药物。
4.一种用于肿瘤诊疗一体化的纳米复合药物的制备方法，其特征在于，化疗药物DOX和光动力药物TMPyP4的加载方法包括：将化疗药物DOX与制备的双面神结构探针Au-I混合三小时以上，反应结束后通过在8000r离心5分钟从溶液中去除过量的药物并分散在Trisbuffer中，得到Au-I-DOX，将TMPyP4与Au-I-DOX探针混合通过在7500rpm下离心5分钟除去游离的TMPyP4，得到Au-I-D/T。
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