通过从光响应聚合物纳米载体的siRNA释放的时空控制预测基因沉默
Summary
我们提出一种新的方法，使用光响应嵌段共聚物更有效的时空控制基因沉默，没有可检测的脱靶效应。此外，可以使用简单的siRNA释放测定法和简单的动力学建模来预测基因表达的变化。
Abstract
需要新的材料和方法来更好地控制需要精确调节基因活性的广泛应用的核酸的结合与释放。特别地，具有改进的基因表达时空控制的新型刺激响应材料将在药物发现和再生医学技术中解锁可翻译平台。此外，增强的控制从材料释放核酸的能力将使得能够开发精简的方法来先验地预测纳米载体功效，从而加速输送载体的筛选。在这里，我们提出了一种通过模块化光响应纳米载体系统预测基因沉默效率和实现基因表达的时空控制的方案。小干扰RNA（siRNA）与mPEG- b-聚（甲基丙烯酸5-（3-（氨基）丙氧基）-2-硝基苄酯）（mPEG- b- P（APNBMA））聚合物与聚rm稳定的纳米载体，可以用光控制，以促进可调谐，开/关siRNA释放。我们概述了使用荧光相关光谱和凝胶电泳的两种互补测定法，用于从模拟细胞内环境的溶液中精确定量siRNA释放。从这些测定中获得的信息被并入到简单的RNA干扰（RNAi）动力学模型中，以预测对各种光刺激条件的动态沉默反应。反过来，这些优化的照射条件允许改进用于时空控制基因沉默的新方案。该方法可以产生具有细胞至细胞分辨率的基因表达中的细胞模式，并且不产生可检测到的脱靶效应。总之，我们的方法提供了一种易于使用的方法来预测基因表达的动态变化并精确控制空间和时间的siRNA活性。这组测定可以容易地适应于测试各种各样的她的刺激反应系统，以解决与生物医学研究和医学中众多应用相关的关键挑战。
Introduction
小干扰RNA（siRNA）通过催化RNAi途径介导转录后基因沉默，该途径对于几乎任何靶基因1具有高度特异性，有效性和可裁剪性。这些有希望的特性已启用siRNA治疗在众多疾病，包括转移性黑素瘤和血友病2,3治疗人类临床试验来推进。然而，重大的交付问题仍然存在，妨碍了翻译4 。特别是，送货车辆必须保持稳定和保护的siRNA从细胞外降解，但也释放有效载荷进入细胞质5。此外，许多RNAi应用需要改进的方法来调节空间和时间6中的基因沉默，这将减少siRNA治疗药物7中的副作用，并使得能够转化应用范围从药物发现的细胞芯片8到再生支架中细胞反应调节的应用9 。这些挑战强调需要新的材料和方法来更好地控制siRNA纳米载体中的结合和释放。
控制siRNA释放和增强时空调节的最有希望的策略之一是使用刺激响应材料10 。例如，响应于改变的氧化还原电位或pH或施加的磁场，超声波或光11 ，已经将各种各样的生物材料设计成具有可变核酸结合亲和力。尽管许多这些系统证明改进了对核酸活性的控制，但由于其瞬时瞬时响应，精确的空间分辨率和易调节性，使用光作为触发器是特别有利的<sup class =“xref”> 12。此外，光敏技术调节基因表达的潜力已经由最先进的诱导型启动子和光遗传调控系统证明;然而，这些系统从许多挑战包括有限的能力来调节内源基因，安全问题，如免疫原性，以及在提供多部件组件13，14，15遭受困难。光响应siRNA的纳米载体是非常适合克服这些缺点，并提供一种更简单，更可靠的方法来时空调节基因表达16，17，18。不幸的是，准确预测所得到的蛋白质敲低反应的方法仍然难以捉摸。
一个关键的挑战是定量评估siRNA释放罕见19,20，甚至当执行这些评估，它们还没有被耦合到的siRNA /蛋白质周转动力学分析。 siRNA的释放量及其持久性/寿命都是所得基因沉默动力学的重要决定因素;因此，缺乏这样的信息是一个重大的断开，排除RNAi 21中剂量反应的准确预测。解决这一挑战将加快制定纳米载体中适当的结构 – 功能关系，并更好地告知生物材料设计22 。此外，这种方法将能够开发出更有效的siRNA给药方案。在试图了解动态响应沉默，几组已经研究RNAi的23，24，25的数学模型。这些框架是成功地提供对siRNA介导的基因表达变化的认识并鉴定速率限制步骤26 。然而，这些模型仅被应用于不能控制siRNA释放的商业基因递送系统（ 例如 ，Lipofectamine和聚乙烯亚胺（PEI）），并且模型的复杂性严重限制了其实用性27 。这些缺点突显出了能够精确调整siRNA释放的新材料与流线型易于使用的预测动力学模型的未满足需求。
我们的方法通过将光敏纳米载体平台与耦合方法整合来量化免费的siRNA和模型RNAi动力学来解决所有这些挑战。特别地，我们的平台的精确控制的siRNA释放28通过两种互补方法进行监测，用于精确定量封装对比结合siRNA。这些测定的实验数据被输入到一个简单的动力学模型，以预测基因沉默效率先验 29 。最后，纳米载体的开/关性质容易被利用以在细胞长度尺度上产生具有空间控制的基因表达中的细胞模式。因此，该方法提供了一种易于适应的方法来控制和预测将受益于细胞行为的时空调节的各种应用中的基因沉默。
Protocol
Representative Results
Discussion
该方法中有几个步骤特别重要。当配制纳米载体，组分添加和混合速度的顺序是影响功效39的两个重要参数。该方案要求在涡旋下将阳离子组分mPEG- b- P（APNBMA）以滴加方式加入到阴离子组分siRNA中。取决于总制剂体积，该混合过程需要3-6秒。为了测试纳米载体是否正确形成，使用诸如动态光散射的技术来测量尺寸分布。所述的mPEG-b -P（APNBMA）/ siRNA的纳米载体与4的N /…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢美国国家卫生研究院国家卫生研究院（NIH）通过授权号P20GM103541的机构发展奖（IDeA）以及拨款号P20GM10344615提供财政支持。本文中的声明不反映NIH的观点。我们还通过生物科学高级技术中心（Bioscience CAT）奖（12A00448），承认特拉华生物技术研究所（DBI）和特拉华州经济发展办公室（DEDO）的财务支持。
Materials
| siRNA | Sigma-Aldrich | SIC001 | non-targeted, universal negative control |
| mPEG-b-P(APNBMA) | synthesized in our lab | N/A | photo-responsive polymer |
| HEPES | Fisher Scientific | BP310-100 | |
| sodium dodecyl sulfate | Sigma-Aldrich | 436143 | |
| rubber gasket | McMaster-Carr | 3788T21 | 0.5 mL thick |
| UV laser | Excelitas Technologies | Omnicure S2000 | collimating lens and 365 nm filter used |
| agarose | Fisher Scientific | BP160-100 | |
| ethidium bromide | Fisher Scientific | BP1302-10 | |
| siRNA labelled with Dy547 | GE Healthcare Dharmacon, Inc. | custom order | fluorophore conjugated to 5’ end of sense strand |
| microscope slide | Fisher Scientific | 12-550-A3 | pre-cleaned glass |
| Secure-Seal Spacer | Life Technologies | S24735 | double-sided adhesive |
| LSM 780 | Carl Zeiss | N/A | confocal microscope |
| ZEN 2010 | Carl Zeiss | N/A | FCS analysis software |
| MATLAB | MathWorks | N/A | programming language |
| NIH/3T3 cells | ATCC | ATCC CRL-1658 | |
| DMEM | Mediatech | 10-013-CV | growth media |
| fetal bovine serum | Mediatech | 35-011-CV | heat-inactivated |
| penicillin-streptomycin | Mediatech | 30-002-CI | |
| 6-well plates | Fisher Scientific | 08-772-1B | |
| Opti-MEM | Life Technologies | 11058021 | transfection media |
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