Name: predicting gene silencing through the spatiotemporal control of sirna release from photo-responsive polymeric nanocarriers
Uploaded: 2017-07-21T15:00:00
Description: Watch this Scientific Journal Video about Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers at JoVE.com

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。

1. siRNA纳米载体的制备<ol><li> 在pH 6.0下，以等体积稀释于20mM 4-（2-羟乙基）哌嗪-1-乙磺酸（HEPES）缓冲液中制备siRNA和mPEG- b- P（APNBMA）的单独溶液。 <ol><li>加入浓度为32μg/ mL至20 mM HEPES溶液的siRNA。 注意：siRNA是非靶向的通用阴性对照序列;然而，siRNA可以设计成靶向任何感兴趣的基因。 </li><li>将mPEG- b- P（APNBMA）聚合物溶解到20mM HEPES溶液中。加入适量的mPEG- b- ...

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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 /...

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。

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">siRNA</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:145px;">SIC001</td>
			<td style="width:195px;">non-targeted, universal negative control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">mPEG-b-P(APNBMA)</td>
			<td style="width:157px;">synthesized in our lab</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">photo-responsive polymer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">HEPES</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP310-100</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">sodium dodecyl sulfate&#160;</td>
			<td style="width:157px;">Sigma-Aldrich</td>
			<td style="width:145px;">436143</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">rubber gasket</td>
			<td style="width:157px;">McMaster-Carr</td>
			<td style="width:145px;">3788T21</td>
			<td style="width:195px;">0.5 mL thick</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">UV laser&#160;</td>
			<td style="width:157px;">Excelitas Technologies</td>
			<td style="width:145px;">Omnicure S2000</td>
			<td style="width:195px;">collimating lens and 365 nm filter used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">agarose</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP160-100</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">ethidium bromide</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">BP1302-10</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">siRNA labelled with Dy547</td>
			<td style="width:157px;">GE Healthcare Dharmacon, Inc.</td>
			<td style="width:145px;">custom order</td>
			<td style="width:195px;">fluorophore conjugated to 5&#8217; end of sense strand</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">microscope slide</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">12-550-A3</td>
			<td style="width:195px;">pre-cleaned glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Secure-Seal Spacer</td>
			<td style="width:157px;">Life Technologies</td>
			<td style="width:145px;">S24735</td>
			<td style="width:195px;">double-sided adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">LSM 780&#160;</td>
			<td style="width:157px;">Carl Zeiss</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">confocal microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">ZEN 2010</td>
			<td style="width:157px;">Carl Zeiss</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">FCS analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">MATLAB</td>
			<td style="width:157px;">MathWorks</td>
			<td style="width:145px;">N/A</td>
			<td style="width:195px;">programming language</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">NIH/3T3 cells&#160;</td>
			<td style="width:157px;">ATCC</td>
			<td style="width:145px;">ATCC CRL-1658</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">DMEM</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">10-013-CV</td>
			<td style="width:195px;">growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">fetal bovine serum</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">35-011-CV</td>
			<td style="width:195px;">heat-inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">penicillin-streptomycin</td>
			<td style="width:157px;">Mediatech</td>
			<td style="width:145px;">&#160;30-002-CI</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">6-well plates</td>
			<td style="width:157px;">Fisher Scientific</td>
			<td style="width:145px;">08-772-1B</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Opti-MEM</td>
			<td style="width:157px;">Life Technologies</td>
			<td style="width:145px;">11058021</td>
			<td style="width:195px;">transfection media</td>
		</tr>
	</tbody>
</table>

predicting gene silencing through the spatiotemporal control of sirna release from photo-responsive polymeric nanocarriers

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。

在制备纳米载体之后，进行siRNA释放测定以通知用于体外转染的照射条件。应用各种剂量的光以确定被释放的siRNA的百分比。第一个测定法使用凝胶电泳将游离的siRNA分子与仍然与聚合物复合/相关的siRNA分子分离。没有用光处理的纳米载体保持稳定并且不释放任何siRNA。随着照射时间的延长，游离siRNA带的荧光强度增加。使用图像分析软件对带强度的定量表明，在20分?...

Watch this Scientific Journal Video about Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers at JoVE.com

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

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。

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

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Research

JoVE Journal

Bioengineering

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

制定通过Nanoprecipitation技术嵌段聚合物纳米粒子

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

科研

生物工程

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

组合合成和高通量的蛋白质释放的聚合物薄膜和纳米粒子库

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

工程双层凝胶控制升序分化

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

的使用<em&gt;前体内</em&gt;钱德勒循环装置，以评估改性聚合物血导管的生物相容性

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

多孔硅微粒进行交付的siRNA治疗的

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Polymeric Microneedle Array Fabrication by Photolithography

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

制造超疏水高分子材料生物医学应用

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

在脉冲激光激发金纳米粒子集成光电响应脂质体及其微泡空化的测量的合成

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...