CN113549132A - 一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用 - Google Patents
一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用，所述制备方法包括以下步骤：将反义寡核苷酸与三联吡啶氯化钌溶液混合，孵育得到DNA‑[Ru(bpy)3]2+溶液；将DNA‑[Ru(bpy)3]2+溶液、含酪氨酸的靶向肽溶液、过硫酸铵溶液混合，进行白光照射，离心后即可得到所述非病毒载体；其具有合成简单，绿色，高效等特点；该非病毒载体可在在基因传递或作为靶向药物中进行应用。
Description
技术领域
本发明属于基因输送载体技术领域，具体涉及一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用。
背景技术
药物治疗涉及多种复杂的机制，作一种智能和适应性系统是目前肿瘤研究和临床实践中的一大挑战，癌细胞可以发展多种防御策略来规避治疗药物的影响。这些防御机制通常与耐药相关蛋白表达水平的改变有关，最终导致抗癌治疗的失败。为应对这一挑战，反义寡核苷酸(ASO)和小干扰RNA (sirna)等是能够抑制耐药相关蛋白的表达以恢复癌细胞的药物敏感性的核酸治疗方法，其已与光动力联合使用，达到加法和协同治疗效果。由于光动力治疗药物和核酸具有本质上的不同，常规光动力治疗和基因组合治疗通常采用一种载体来装载光学药物和核酸治疗药物，每个组分分别发挥各自的作用。尽管取得了巨大的进步，但仍然存在局限性。
发明内容
本发明的目的在于提供一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用，本发明通过反义寡核苷酸中的dsDNA双螺旋结构插入[Ru(bpy)3]Cl2形成DNA-[Ru(bpy)3]2+，在白光照射下，DNA-[Ru(bpy)3]2+催化含酪氨酸的靶向肽YY-12共价交联，自组装形成肽-核酸纳米球(RGD-NS)非病毒载体，其具有合成简单，绿色，高效等特点；该非病毒载体可在在基因传递或作为靶向药物中进行应用。
为实现上述目的，本发明采取的技术方案如下：
一种基于DNA-[Ru(bpy)3]2+催化短肽共价组装形成非病毒载体的制备方法，所述制备方法包括以下步骤：
(1)将反义寡核苷酸与三联吡啶氯化钌溶液混合，孵育得到DNA-[Ru(bpy)3]2+溶液；
(2)将步骤(1)得到的DNA-[Ru(bpy)3]2+溶液、含酪氨酸的靶向肽溶液、过硫酸铵溶液混合，进行白光照射，离心后即可得到所述非病毒载体；
所述含酪氨酸的靶向肽的氨基酸序列为YYRRVRRRGDYY；其中，Y为酪氨酸，RVRR为具有弗林酶切割位点多肽，RGD是可与αvβ3整合素和sigma受体过表达的肿瘤可以主动结合的序列肽。
步骤(1)中，所述反义寡核苷酸的制备方法为：将DNA S1溶液和DNA S2溶液混合，90℃退火10min，并在4～10℃静置至形成双链DNA结构；
所述DNA S1基因序列为：
3’-GGA TTG GAG TTC CTC CAG CGT GCG CCA TCC TTC CCA TCC TCCTCC-5’；划横线的部分为可降低抗凋亡蛋白Bcl-2表达的ASO的序列。
所述DNA S2基因序列为：3’-GAG GAA CTC CAA TCC-5’。
所述DNA S1、DNA S2的摩尔比为1:1。
步骤(1)中，反应体系中，反义寡核苷酸、三联吡啶氯化钌的摩尔比为1：800～1200，优选为1:1000。
步骤(1)中，所述孵育的条件为34～37℃孵育6.5～7.5h，优选为37℃孵育7.0h。
步骤(2)中，DNA-[Ru(bpy)3]2+溶液、短肽溶液、过硫酸铵溶液的体积之比为1：0.8～1.2：2.5～3.2，优选为1:1:3；所述短肽溶液的浓度为0.5～1.5mg/mL，优选为1.0mg/mL；所述过硫酸铵溶液的浓度为8～12mM，优选为10mM。
步骤(2)中；所述照射的时间为5～10min，优选为6min。
步骤(2)中，使用海门其林贝尔GL-800白光透射仪进行白光照射。
按照本发明所述的制备方法制备得到的非病毒载体，其为平均粒径200nm的纳米球，且纳米球中的C、Ru、P等元素均匀分布，所述非病毒载体实现了将核酸包封在交联体内，实现对核酸的运载。
本发明还提供了所述的非病毒载体在基因传递或作为靶向药物中的应用，通过非病毒载体实现了对核酸的包封，其在弗林酶的作用下可释放出包封的核酸，进而实现基因的传递及疾病的靶向治疗。
本发明还提供了一种可释放所述非病毒载体中的DNA的方法，将弗林酶加入到所述非病毒载体中进行切割。
具体为：将弗林酶溶液加入到所述非病毒载体中，消化1h，加入EDTA终止弗林酶的活性。在白光照射下，将酶解产物通过ESR检测羟基自由基。
所述弗林酶溶液的制备方法为：将弗林酶溶解在pH＝7.4的tris-HCl溶液中得到质量浓度为0.1％弗林酶溶液。
本发明提供的基于DNA-[Ru(bpy)3]2+催化短肽共价组装成非病毒载体的制备方法中，首先，利用反义寡核苷酸的dsDNA双螺旋结构插入[Ru(bpy)3]Cl2形成DNA-[Ru(bpy)3]2+，在白光照射下，DNA-[Ru(bpy)3]2+催化含酪氨酸的靶向肽形成含有酪氨酸自由基，两个酪氨酸自由基共价交联，自组装形成肽-核酸纳米球(RGD-NS)非病毒载体，该载体是直径约为200nm肽-核酸纳米球非病毒载体。该非病毒载体可实现对于DNA的良好包封，并在弗林酶的作用下水解，释放DNA-[Ru(bpy)3]2+，其中被保护的核酸ASO(Anti-sense single-strandedDNA oligonucleotide)，其可通过碱基互补配对与靶标的mRNA结合，使得靶标RNA被RNaseH切割引发基因沉默，提高细胞对于药物的敏感性，另一方面释放的[Ru(bpy)3]2+在光照下产生羟基自由基，两者协同进行抗肿瘤治疗。该方法具有合成简单，高效且是毒性低等特点。
附图说明
图1为基于DNA-[Ru(bpy)3]2+催化短肽共价组装的非病毒载体的制备方法示意图；
图2为实施例1中的肽-核酸纳米球非病毒载体的形态和特性，其中，a)肽-核酸纳米球非病毒载体的SEM图；b)肽-核酸纳米球非病毒载体的TEM图；c)为肽-核酸纳米球非病毒载体的元素分布图像；d)肽-核酸纳米球的粒径分布图；
图3为(a)dsDNA与[Ru(bpy)3]Cl2孵育生成DNA-[Ru(bpy)3]2+复合物荧光图；(b)短肽溶液与过硫酸铵及DNA-[Ru(bpy)3]Cl2孵育生成纳米复合物过程中Zeta电势变化图；
图4为短肽溶液与过硫酸铵及DNA-[Ru(bpy)3]2+孵育生成荧光图及(b)不同时间下测试体系的紫外吸收图；
图5为纳米复合物及纳米复合物经弗林酶处理后的产物在200mw·cm-2的532nm光照射后产生羟基自由基的图；
图6为PBS、YY-12、DNA-[Ru(bpy)3]2+、肽-核酸纳米球分别处理在A549细胞，经AM/PI染色后的荧光成像图。
具体实施方式
下面结合实施例对本发明进行详细说明。
实施例中的各溶液是通过以下方法制备的：
DNA S1溶液和DNA S2溶液：分别将通过将DNA S1、DNA S2溶解在pH＝7.4的10mM的Tris-HCl缓冲溶液中得到；
[Ru(bpy)3]Cl2溶液：将三联吡啶氯化釕溶解在超纯水溶液中得到；
短肽溶液：是将短肽溶解在超纯水中得到；
过硫酸铵溶液：将过硫酸铵溶解在超纯水中得到的。
实施例1
一种基于DNA-[Ru(bpy)3]2+催化短肽共价组装形成非病毒载体的制备方法，所述制备方法包括以下步骤：
(1)将等体积的浓度均为2μM的DNA S1溶液和DNA S2溶液加热到90℃，10分钟后取出，冷却至室温，随后保存在温度为4～10℃的冰箱上层至形成dsDNA结构，得到dsDNA溶液；
所述DNA S1的基因序列为：3’-GGA TTG GAG TTC CTC CAG CGT GCG CCA TCC TTC CCA TCCTCC TCC-5’；划横线的部分为可降低抗凋亡蛋白Bcl-2表达的ASO的序列，如果将其替换为可治疗其他疾病的核酸序列，同样可实现其他疾病的靶向治疗；
所述DNA S2的基因序列为：3’-GAG GAA CTC CAA TCC-5’；
(2)将1mL 2mM[Ru(bpy)3]Cl2溶液加入1mL的dsDNA溶液中，37℃孵育7h，形成DNA-[Ru(bpy)3]2+溶液；
(3)将0.2mL 1mg/ml短肽(YY-12)溶液与0.2mL DNA-[Ru(bpy)3]2+溶液及0.6mL10mM过硫酸铵溶液混合混匀，然后白光下照射6min，使用海门其林贝尔GL-800白光透射仪进行白光照射，得到肽-核酸纳米球(RGD-NS)溶液；所述短肽的氨基酸序列为YYRVRRRGDYY，其中Y为酪氨酸，RVRR为具有弗林酶切割位点多肽，RGD是一种靶向为多肽；
本实施例制备得到的非病毒载体的TEM、SEM、mapping图如图2所示，从其SEM、TEM，DLS图中可以看出其约为200nm肽-核酸纳米球。进一步对元素映射图像进行了探究，C、Ru和P等元素的也显示均匀分布。
实施例2
其他同实施例1，在进行步骤(2)时测试[Ru(bpy)3]Cl2、DNA-[Ru(bpy)3]2+的荧光光谱，如图3a)所示，生成DNA-[Ru(bpy)3]2+时625nm特征荧光峰强度的增加，证明DNA-[Ru(bpy)3]2+小分子的形成；同时在进行步骤(2-4)时进行Zeta电势的测量，如图3b)所示，当向[Ru(bpy)3]Cl2溶液中添加dsDNA溶液制备DNA-[Ru(bpy)3]2+时，DNA-[Ru(bpy)3]2+的Zeta电位绝对值变得比dsDNA小，这是由于[Ru(bpy)3]2+带正电荷，也进一步证明了[Ru(bpy)3]2+在dsDNA里的成功嵌入得到了DNA-[Ru(bpy)3]2+；当DNA-[Ru(bpy)3]2+与YY-12混合经光照射得到非病毒载体后，zeta电位进一步变的更正，这可能是由于酪氨酸共价交联聚精氨酸更多暴露在球外所致。
实施例3
其他同实施例1，在进行步骤(4)时测试YY-12、及反应得到的非病毒载体的荧光光谱，如图3a)所示，生成的肽-核酸纳米球的荧光峰为410nm，证明非病毒载体的形成；反应物混合均匀后，照射不同时间下取样测试体系的紫外吸收图，测试时间截止在600s，6min后峰值趋于平缓不再上升，此时的紫外吸光度值达到最大，也证明了短肽共价交联的成功，成功制备得到了非病毒载体。
实施例4
一种可释放所述非病毒载体中的DNA的方法，包括以下步骤：
将50μL质量浓度为0.1％弗林酶溶液加入到实施例1制备得到的肽-核酸纳米球溶液中，消化1h，加入EDTA终止弗林酶的活性。在532nm白光照射下，通过ESR分别检测非病毒载体、酶解产物中的羟基自由基。结果如图5所示，结果显示经弗林酶处理的溶液，有明显的羟基自由基信号；而对于相同浓度、相同辐照时间的肽-核酸纳米球溶液，信号微弱几乎可忽略不计。再次表明本发明制备得到的非病毒载体肽-核酸纳米球是一种弗林酶响应的纳米载体。
实施例5
为了研究肽-核酸纳米球的抗癌作用，进行了荧光成像展示。
将实施例1制备的非病毒载体肽-核酸纳米球中DNA的浓度调整为原来的10倍而其他原料的浓度保持不变进行包裹加入A549细胞中，用AM-PI对活/死细胞染色来进一步证实肽-核酸纳米球对A549细胞的光动力及基因治疗作用，以同等浓度的PBS、YY-12、NS为对照。共聚焦显微镜图像清楚地表明，在532nm光辐射下，经RGD-NS处理的细胞几乎全部凋亡，而DNA-[Ru(bpy)3]2+处理细胞的仅有部分细胞凋亡，这表明肽-核酸纳米球(RGD-NS)是一个良好的非病毒纳米载体。
上述实验中，肽-核酸纳米球与A549细胞共孵育，A549细胞中的高表达的弗林酶切割肽-核酸纳米球，释放出DNA-[Ru(bpy)3]2+复合物，其中与靶标的mRNA结合，使得靶标RNA被RNase H切割引发基因沉默，恢复癌细胞对于药物的敏感性，同时用白光照射，[Ru(bpy)3]2+产生羟基自由基损伤细胞，两者协同作用使得癌细胞凋亡。
上述参照实施例对一种基于三联吡啶釕催化短肽共价组装形成的非病毒载体及其制备方法和应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。
Claims (10)
1.一种基于DNA-[Ru(bpy)3]2+催化短肽共价组装形成非病毒载体的制备方法，其特征在于，所述制备方法包括以下步骤：
(1)将反义寡核苷酸与三联吡啶氯化钌溶液混合，孵育得到DNA-[Ru(bpy)3]2+溶液；
(2)将步骤(1)得到的DNA-[Ru(bpy)3]2+溶液、含酪氨酸的靶向肽溶液、过硫酸铵溶液混合，进行白光照射，离心后即可得到所述非病毒载体。
2.根据权利要求1所述的制备方法，其特征在于，所述含酪氨酸的靶向肽的氨基酸序列为YYRRVRRRGDYY。
3.根据权利要求1所述的制备方法，其特征在于，步骤(1)中，所述反义寡核苷酸的制备方法为：将DNA S1溶液和DNA S2溶液混合，90℃退火10min，并在4～10℃静置至形成双链DNA结构；
所述DNA S1基因序列为：
3’-GGA TTG GAG TTC CTC CAG CGT GCG CCA TCC TTC CCA TCC TCC TCC-5’；
所述DNA S2基因序列为：3’-GAG GAA CTC CAA TCC-5’。
4.根据权利要求1-3任意一项所述的制备方法，其特征在于，步骤(1)中，反应体系中，反义寡核苷酸、三联吡啶氯化钌的摩尔比为1：800～1200。
5.根据权利要求1-3任意一项所述的制备方法，其特征在于，步骤(1)中，所述孵育的条件为34～37℃孵育6.5～7.5h。
6.根据权利要求1-3任意一项所述的制备方法，其特征在于，步骤(2)中，DNA-[Ru(bpy)3]2+溶液、含酪氨酸的靶向肽溶液、过硫酸铵溶液的体积之比为1：0.8～1.2：2.5～3.2；所述含酪氨酸的靶向肽溶液的浓度为0.5～1.5mg/mL；所述过硫酸铵溶液的浓度为8～12mM。
7.根据权利要求1-3任意一项所述的制备方法，其特征在于，步骤(2)中，所述照射的时间为5～10min。
8.如权利要求1-7任意一项所述的制备方法制备得到的非病毒载体。
9.根据权利要求8所述的非病毒载体在基因传递或作为靶向药物中的应用。
10.一种可释放权利要求9所述的非病毒载体中的DNA的方法，其特征在于，将弗林酶加入到所述非病毒载体中进行切割。
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