Name: predicting gene silencing through the spatiotemporal control of sirna release from photo-responsive polymeric nanocarriers
Uploaded: 2017-07-21T15:00:00.000+00:00
Duration: 11 min 53 s
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- P（APNBMA）制成220μg/ mL溶液，使得N / P比（N，mPEG- b- P（APNBMA）上的胺基团; siRNA上的P，磷酸基团）是4。 注意:mPEG- b- P（APNBMA）聚合物的合成方案在其他地方报道30 。 </li></ol></li><li>将mPEG- b- P（APNBMA）溶液逐滴添加至相等的体积e的siRNA溶液，同时在涡流机上轻轻混合。加入聚合物后继续涡旋30秒。在室温下将样品在黑暗中孵育30分钟。 </li></ol> 2.使用凝胶电泳测量siRNA释放<ol><li> 根据步骤1.1-1.2制备纳米载体，并缩放体积以适应所需的样品数量。 </li><li>将纳米载体与十二烷基硫酸钠（SDS）混合。 <ol><li>制备1 mg / mL SDS的水溶液。等分出产生S / P比（S，SDS上的硫酸根; siRNA上的磷酸酯基团）为15的溶液所需的SDS溶液的量。 注意:如果polyplex溶液含有1μgsiRNA，则必须加入13μgSDS以达到15的S / P比。 </li><li>将SDS溶液滴加到每个纳米载体溶液中，同时轻轻混合在涡流机上。 SDS添加后继续涡旋30秒。 </li><li&gt;以3,000 xg离心5 s。在室温下将样品在黑暗中孵育30分钟。 </li></ol></li><li>校准并设置具有365nm滤光片的UV激光器至200W / m的强度。确保从样品溶液的底部就座的位置测量光强度。 </li><li> 将纳米载体/ SDS溶液装载到由橡胶垫片分隔开的玻璃片组成的玻璃室中。 <ol><li>在7:3（v / v）乙醇/水溶液中预先洗涤玻片在水中，并彻底干燥。将一个孔（约2 x 3厘米长方形）切成橡胶垫圈。将橡胶垫圈放在玻璃滑块上。 </li><li>将纳米载体/ SDS溶液吸取到橡胶垫片孔内的玻璃载玻片上。将过量的溶液（剩余20μL）装入玻璃载片上，同时避免与橡胶垫圈接触。 注意:有些液体会在后续步骤中丢失。 </li><li>放第二块玻璃在滑动垫片的顶部滑动。为了避免产生气泡，首先将滑块的一端放下，然后缓慢降低另一端。 </li><li>将夹子夹在玻璃室的每一侧以将其封闭。 </li></ol></li><li>使用具有365nm滤光器的UV激光以200W / m的强度将样品辐照所需的时间长度（ 例如 ，0-60分钟）。拆下夹子夹并打开腔室。 </li><li>将25μL经辐射的纳米载体/ SDS样品吸取到微量离心管中。将溶液在室温下在黑暗中孵育30分钟。 </li><li>准备根据标准方案31在Tris /硼酸盐/ EDTA（TBE）缓冲溶液中用0.5μg/ mL溴化乙锭预染色的2重量％琼脂糖凝胶。制备由3:7（v / v）甘油/水组成的加载缓冲液。 </li><li>将5μL加载缓冲溶液加入到每个纳米载体/ SDS样品的25μL中。在黑暗中孵育样品室温10分钟。 </li><li>将每个纳米载体/ SDS样品加入到2％琼脂糖凝胶中。在100 V的黑暗中运行凝胶30 min。使用凝胶成像系统对溴化乙锭过滤器进行成像。保存凝胶图像文件，并进行步骤2.10进行带强度量化。确保频带强度足够亮以清晰可视，但不要太亮，信号饱和。 </li><li> 使用公开的ImageJ软件32来量化频带强度。 <ol><li>使用ImageJ的ROI工具，通过在每个条带周围绘制一个矩形来确定每条泳道中游离siRNA条带的荧光强度。绘制每个通道的强度曲线，并通过在强度曲线上绘制水平线并点击封闭区域内的追踪棒来整合曲线下面积。 </li><li>计算每个车道的相对强度，除以下面积的面积通过siRNA阳性对照曲线下的面积（不添加mPEG- b- P（APNBMA）并且不添加SDS）的每个样品的rve。报告每个样品的标准化带强度释放的siRNA百分比。 </li></ol></li></ol> 3.使用荧光相关光谱（FCS）测量siRNA释放<ol><li>获得在有义链的5&#39;末端用单个荧光团标记的siRNA。 注意:可以购买预先与所需位置缀合的标记物进行退火的siRNA。荧光团应该是光稳定的并且吸收/发射在450和750nm之间以避免UV光淬灭和与mPEG- b- P（APNBMA）的能量转移。 </li><li>使用标记的siRNA根据步骤1.1-1.2制备纳米载体。缩放体积以适应所需的样品数量。 </li><li>在SDS中孵育溶液，并按照步骤2.2-2.6照射所需的时间长度。 </li><li> PreparFCS样品室。 <ol><li>用7:3（v / v）乙醇/水溶液洗涤玻片，并使用擦拭物和空气流将玻璃完全干燥。 </li><li>从双面粘合隔离物上取下纸张以露出双面粘合隔离物。将间隔件连接到玻璃盖玻片上。 </li><li>将纳米载体/ SDS溶液从粘合间隔物移到孔的中间的盖板上。 </li><li>将玻璃滑块放在盖玻片的顶部。推上玻璃滑块，以确保玻璃滑块和盖玻片安装良好并形成密封。 </li></ol></li><li> 使用共焦显微镜进行FCS测量33 。使用数值孔径为1.2的40X水浸式复消色差物镜。使用适当的激发激光通道（488 nm），每个样品34采集至少30次测量值为10 s。确保激光强度和检测器对准保持不变对于每个样品也是一样的。 <ol><li>除实验样品外，测量对照包括:无标记siRNA的空白样品;和具有标记的siRNA但不含mPEG- b- P（APNBMA）的游离siRNA样品。 </li></ol></li><li> 使用FCS专用软件分析数据。通过确定在没有纳米载体通过共焦体积29的时间内的稳定计数率来识别每个样品的基线计数率。 <ol><li>从每个样本基准计数率值中减去空白样本的计数率。将得到的值归一化为游离siRNA对照，以计算游离siRNA 35的百分比。 </li></ol></li></ol> 4.动力学建模以预测基因沉默<ol><li> 使用简单的普通微分方程组建立数学编程语言脚本来预测基因沉默29。 注意:可根据要求提供脚本。 <ol><li>将普通微分方程组写为: <img alt="公式1" src="/files/ftp_upload/55803/55803eq1.jpg" /> （1） <img alt="公式2" src="/files/ftp_upload/55803/55803eq2.jpg" /> （2） <img alt="公式3" src="/files/ftp_upload/55803/55803eq3.jpg" /> （3） 注意:对于等式1-3，术语k mRNA ，k siRNA和k prot是分别产生mRNA，siRNA和蛋白质的速率常数。术语k m，deg ，k s，deg和k p，deg分别是mRNA，siRNA和蛋白质降解的速率常数。降解速率常数基于组分半衰期计算，生产速率常数适合于确保在不存在o时达到mRNA和蛋白质稳态值f siRNA。 <ol><li>确定感兴趣的基因的mRNA和蛋白质的半衰期，如参考文献36或文献所述的实验（参见讨论 ）。此外，确定细胞系的倍增时间。将这些值输入到适当的降解率表达式中。 </li><li>调整生产速率常数，使得如果不引入siRNA，基因表达水平保持稳定在标准化值100。具体来说，将[siRNA]设定为零，并且改变k mRNA ，k siRNA和k prot生成速率常数的值，直到[mRNA]和[蛋白质]保持在初始标准化值的1％的1％内，持续时间为模拟。 </li><li>使用从前述凝胶电泳和FCS测定法释放的siRNA的相对量作为估计值，调整脚本中siRNA的初始相对浓度。具体来说，[siRNA]与释放的siRNA的相对量成比例，其值为100，对应于最大量29 。 </li></ol></li></ol></li></ol>细胞培养和体外 siRNA递送<ol><li> 根据供应商的方案培养NIH / 3T3鼠胚胎成纤维细胞。 <ol><li>在生长培养基（Dulbecco&#39;s Modified Eagle培养基（DMEM）中培养细胞，补充有10％热灭活的胎牛血清和1％青霉素 - 链霉素）。保持细胞在37℃在5％CO 2的加湿环境中。 </li></ol></li><li> 将细胞种植在6孔组织培养处理板中。 <ol><li>按照供应商推荐的传代培养程序。使用血细胞计数器计数细胞。将补充生长培养基中的细胞稀释至75,000个细胞/ mL的浓度。 </li><li>加入2 mL细胞悬浮液（75,000个细胞s / mL）到6孔板的每个孔。让细胞在培养箱中粘附并恢复24小时。 </li></ol></li><li>通过用磷酸盐缓冲盐水（PBS）洗涤并向每个孔中加入1.5mL无血清和无抗生素的转染培养基（参见材料表 ）来准备转染细胞。 </li><li>根据步骤1.1-1.2制备siRNA纳米载体。向每个孔中加入25μL含有30pmol siRNA的纳米载体溶液。轻轻移动媒体上下混合。将细胞置于培养箱中3小时。 </li><li>取出转染培养基，并用PBS清洗各孔。加入1 mL补充生长培养基，将细胞置于培养箱中30分钟。 </li><li>为了制备细胞用光刺激治疗，去除补充的生长培养基。向每个孔中加入1mL转染培养基（无酚红）。 注意:确保转染介质不含酚红。 </li><li>校准并设置具有365nm滤光片的UV激光器至200W / m的强度。确保从单元板底部的座位位置测量光强度。 </li><li>将细胞置于37℃的热板上。取下电池板盖。使用具有365nm滤光片的UV激光以200Wm -2的强度从板上方照射细胞达所需时间（长达20分钟）。 </li><li> 取出转染培养基，加入2 mL补充生长培养基。置于培养箱中直至进一步分析（ 例如 ，qPCR为24 h，Western blotting为48 h）。 <ol><li>使用各种技术测量基因表达的变化，如Western blotting 37和qPCR。 38对于基因的可见信号，例如GFP，使用荧光显微镜29。 注意:由于其易用性和准确性，建议使用这些技术在量化基因表达</li></ol></li></ol> 6.控制基因沉默在时空的方式<ol><li>根据步骤5.1-5.7的培养，种子和转染细胞。 </li><li> 准备一个完全阻挡365nm光并使反射最小化的光掩模。 注意:在这种情况下，使用10 x 10厘米的铝箔和黑色建筑纸分别阻挡光并减少反射。将铝箔和建筑纸胶合在一起以形成单个单元。 <ol><li>将所需的形状切割/打孔/加工到光掩模中。例如，使用锋利边缘的刀片和打孔机分别在光掩模中形成直线图案（〜5cm长）和圆形图案（〜7mm直径）。 </li></ol></li><li>将光掩模胶合到6孔板的底部，图案集中在包含具有防反射侧的孔的孔下（ 例如 ，黑色c打印纸）面对板。确保胶水不放置在图案边缘（在〜3 mm内）附近。 </li><li>设置大约25厘米的两个环架，并将平台连接到每个环架上，使平台的高度相等。通过将平板固定在平台的顶部，将两个支架之间的悬挂板悬挂在两个支架之间。确保平板是平的。 </li><li>使用具有365nm滤光片的UV激光以200W / m 2的强度从样品下方照射细胞所需的时间（长达20分钟）。 </li><li>取出转染培养基，加入2 mL补充生长培养基。置于培养箱中至少恢复24小时。使用荧光显微镜对细胞进行成像，如29所述。 </li></ol>

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	<li>Forbes, D. C., Peppas, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+delivery+of+small+RNA+and+DNA.">Oral delivery of small RNA and DNA.</a> J Control Release. 162 (2), 438-445 (2012).</li><li>Davis, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+RNAi+in+humans+from+systemically+administered+siRNA+via+targeted+nanoparticles.">Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles.</a> Nature. 464 (7291), 1067-1070 (2010).</li><li>Bouchie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Companies+in+footrace+to+deliver+RNAi.">Companies in footrace to deliver RNAi.</a> Nat Biotechnol. 30 (12), 1154-1157 (2012).</li><li>Burke, P. A., Pun, S. H., Reineke, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+Polymeric+Delivery+Systems+Amidst+a+Nucleic+Acid+Therapy+Renaissance.">Advancing Polymeric Delivery Systems Amidst a Nucleic Acid Therapy Renaissance.</a> ACS Macro Lett. 2 (10), 928-934 (2013).</li><li>Gooding, M., Browne, L. P., Quinteiro, F. M., Selwood, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=siRNA+Delivery:+From+Lipids+to+Cell-penetrating+Peptides+and+Their+Mimics.">siRNA Delivery: From Lipids to Cell-penetrating Peptides and Their Mimics.</a> Chem Biol Drug Des. 80 (6), 787-809 (2012).</li><li>Motta-Mena, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optogenetic+gene+expression+system+with+rapid+activation+and+deactivation+kinetics.">An optogenetic gene expression system with rapid activation and deactivation kinetics.</a> Nat Chem Biol. 10 (3), 196-202 (2014).</li><li>Wang, X., Chen, X., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+gene+expression+by+a+light-switchable+transgene+system.">Spatiotemporal control of gene expression by a light-switchable transgene system.</a> Nat Methods. 9 (3), 266-269 (2012).</li><li>Ziauddin, J., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays+of+cells+expressing+defined+cDNAs.">Microarrays of cells expressing defined cDNAs.</a> Nature. 411 (6833), 107-110 (2001).</li><li>Saltzman, W. M., Olbricht, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+drug+delivery+into+tissue+engineering.">Building drug delivery into tissue engineering.</a> Nat Rev Drug Discov. 1 (3), 177-186 (2002).</li><li>Mura, S., Nicolas, J., Couvreur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-responsive+nanocarriers+for+drug+delivery.">Stimuli-responsive nanocarriers for drug delivery.</a> Nat Mater. 12 (11), 991-1003 (2013).</li><li>Shim, M. S., Kwon, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-responsive+polymers+and+nanomaterials+for+gene+delivery+and+imaging+applications.">Stimuli-responsive polymers and nanomaterials for gene delivery and imaging applications.</a> Adv Drug Delivery Rev. 64 (11), 1046-1058 (2012).</li><li>Kelley, E. G., Albert, J. N. L., Sullivan, M. O., Epps, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-responsive+copolymer+solution+and+surface+assemblies+for+biomedical+applications.">Stimuli-responsive copolymer solution and surface assemblies for biomedical applications.</a> Chem Soc Rev. 42 (17), 7057-7071 (2013).</li><li>Weber, W., Fussenegger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+biomedical+applications+of+synthetic+biology.">Emerging biomedical applications of synthetic biology.</a> Nat Rev Genet. 13 (1), 21-35 (2012).</li><li>Konermann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+control+of+mammalian+endogenous+transcription+and+epigenetic+states.">Optical control of mammalian endogenous transcription and epigenetic states.</a> Nature. 500 (7463), 472-476 (2013).</li><li>Shimizu-Sato, S., Huq, E., Tepperman, J. M., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-switchable+gene+promoter+system.">A light-switchable gene promoter system.</a> Nat Biotechnol. 20 (10), 1041-1044 (2002).</li><li>Huschka, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Silencing+by+Gold+Nanoshell-Mediated+Delivery+and+Laser-Triggered+Release+of+Antisense+Oligonucleotide+and+siRNA.">Gene Silencing by Gold Nanoshell-Mediated Delivery and Laser-Triggered Release of Antisense Oligonucleotide and siRNA.</a> ACS Nano. 6 (9), 7681-7691 (2012).</li><li>Li, H. -. J., Wang, H. -. X., Sun, C. -. Y., Du, J. -. Z., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-detachable+nanoparticles+based+on+a+light-responsive+amphiphile+for+enhanced+siRNA+delivery.">Shell-detachable nanoparticles based on a light-responsive amphiphile for enhanced siRNA delivery.</a> R Soc Chem Adv. 4 (4), 1961-1964 (2014).</li><li>Braun, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-Activated+Gene+Silencing+via+Gold+Nanoshell-siRNA+Conjugates.">Laser-Activated Gene Silencing via Gold Nanoshell-siRNA Conjugates.</a> ACS Nano. 3 (7), 2007-2015 (2009).</li><li>Gilleron, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+analysis+of+lipid+nanoparticle-mediated+siRNA+delivery,+intracellular+trafficking+and+endosomal+escape.">Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape.</a> Nat Biotechnol. 31 (7), 638-646 (2013).</li><li>Wittrup, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+lipid-formulated+siRNA+release+from+endosomes+and+target+gene+knockdown.">Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown.</a> Nat Biotechnol. 33 (8), 870-876 (2015).</li><li>Roth, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurements+and+rational+materials+design+for+intracellular+delivery+of+oligonucleotides.">Quantitative measurements and rational materials design for intracellular delivery of oligonucleotides.</a> Biotechnol Prog. 24 (1), 23-28 (2008).</li><li>Mao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+polyethylene+glycol+chain+length+on+the+physicochemical+and+biological+properties+of+poly(ethylene+imine)-graft-poly(ethylene+glycol)+block+copolymer/SiRNA+polyplexes.">Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes.</a> Bioconjugate Chem. 17 (5), 1209-1218 (2006).</li><li>Raab, R. M., Stephanopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+gene+silencing+by+RNA+interference.">Dynamics of gene silencing by RNA interference.</a> Biotechnol Bioeng. 88 (1), 121-132 (2004).</li><li>Cuccato, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+RNA+interference+in+mammalian+cells.">Modeling RNA interference in mammalian cells.</a> BMC Systems Biology. 5, 1 (2011).</li><li>Varga, C. M., Hong, K., Lauffenburger, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+synthetic+gene+delivery+vector+design+properties.">Quantitative analysis of synthetic gene delivery vector design properties.</a> Mol Ther. 4 (5), 438-446 (2001).</li><li>Chen, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+comparison+of+intracellular+unpacking+kinetics+of+polyplexes+by+a+model+constructed+from+quantum+Dot-FRET.">Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum Dot-FRET.</a> Mol Ther. 16 (2), 324-332 (2008).</li><li>Bartlett, D. W., Davis, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+kinetics+of+siRNA-mediated+gene+silencing+from+live-cell+and+live-animal+bioluminescent+imaging.">Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging.</a> Nucleic Acids Res. 34 (1), 322-333 (2006).</li><li>Foster, A. A., Greco, C. T., Green, M. D., Epps, T. H., Sullivan, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Mediated+Activation+of+siRNA+Release+in+Diblock+Copolymer+Assemblies+for+Controlled+Gene+Silencing.">Light-Mediated Activation of siRNA Release in Diblock Copolymer Assemblies for Controlled Gene Silencing.</a> Adv Healthc Mater. 4 (5), 760-770 (2015).</li><li>Greco, C. T., Epps, T. H., Sullivan, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+Design+of+Polymer+Nanocarriers+to+Spatiotemporally+Control+Gene+Silencing.">Mechanistic Design of Polymer Nanocarriers to Spatiotemporally Control Gene Silencing.</a> ACS Biomater Sci Eng. 2 (9), 1582-1594 (2016).</li><li>Green, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catch+and+release:+photocleavable+cationic+diblock+copolymers+as+a+potential+platform+for+nucleic+acid+delivery.">Catch and release: photocleavable cationic diblock copolymers as a potential platform for nucleic acid delivery.</a> Polym Chem. 5 (19), 5535-5541 (2014).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Marquer, C., Leveque-Fort, S., Potier, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Lipid+Raft+Partitioning+of+Fluorescently-tagged+Probes+in+Living+Cells+by+Fluorescence+Correlation+Spectroscopy+(FCS).">Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS).</a> J Vis Exp. (62), (2012).</li><li>Staaf, E., Bagawath-Singh, S., Johansson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Diffusion+in+Plasma+Membranes+of+Primary+Lymphocytes+Measured+by+Fluorescence+Correlation+Spectroscopy.">Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy.</a> J Vis Exp. (120), e54756 (2017).</li><li>Buyens, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+disassembly+of+siRNA+polyplexes+in+serum+is+crucial+for+predicting+their+biological+efficacy.">Monitoring the disassembly of siRNA polyplexes in serum is crucial for predicting their biological efficacy.</a> J Control Release. 141 (1), 38-41 (2010).</li><li>Eden, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+Half-Life+Dynamics+in+Living+Human+Cells.">Proteome Half-Life Dynamics in Living Human Cells.</a> Science. 331 (6018), 764-768 (2011).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad. Sci U S A. 76 (9), 4350-4354 (1979).</li><li>Wittwer, C. T., Herrmann, M. G., Moss, A. A., Rasmussen, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+Fluorescence+Monitoring+of+Rapid+Cycle+DNA+Amplification.">Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification.</a> Biotechniques. 54 (6), 314-320 (2013).</li><li>Cho, S. K., Dang, C., Wang, X., Ragan, R., Kwon, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixing-sequence-dependent+nucleic+acid+complexation+and+gene+transfer+efficiency+by+polyethylenimine.">Mixing-sequence-dependent nucleic acid complexation and gene transfer efficiency by polyethylenimine.</a> Biomater Sci. 3 (7), 1124-1133 (2015).</li><li>Liu, Y. M., Reineke, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(glycoamidoamine)s+for+gene+delivery:+Stability+of+polyplexes+and+efficacy+with+cardiomyoblast+cells.">Poly(glycoamidoamine)s for gene delivery: Stability of polyplexes and efficacy with cardiomyoblast cells.</a> Bioconjugate Chem. 17 (1), 101-108 (2006).</li><li>Schwanhausser, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+quantification+of+mammalian+gene+expression+control.">Global quantification of mammalian gene expression control.</a> Nature. 473 (7347), 337-342 (2011).</li><li>Greco, C. T., Muir, V. G., Epps, T. H., Sullivan, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+tuning+of+siRNA+dose+response+by+combining+mixed+polymer+nanocarriers+with+simple+kinetic+modeling.">Efficient tuning of siRNA dose response by combining mixed polymer nanocarriers with simple kinetic modeling.</a> Acta Biomater. 50, 407-416 (2017).</li></ol>

作者宣称他们没有竞争的经济利益。

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。

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

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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 ...

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7.3K 次观看.  University of Delaware. 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。 

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

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

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

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 ...

科研

生物工程

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

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 ...

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

生物学

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

发育生物学

Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic porous silicon nanoparticles (F-pSiNPs) have recently demonstrated high in vivo gene silencing efficacy due to its high oligonucleotide loading capacity and unique cellular uptake pathway that avoids endocytosis. The synthesis of F-pSiNPs is a multi-step process that includes: (1) loading and sealing of oligonucleotide payloads in the silicon pores; (2) simultaneous coating and sizing of fusogenic lipids around the porous silicon cores; and (3) conjugation of targeting peptides and washing to remove excess oligonucleotide, silicon debris, and peptide. The particle&#8217;s size uniformity is characterized by dynamic light scattering, and its core-shell structure may be verified by transmission electron microscopy. The fusogenic uptake is validated by loading a lipophilic dye, 1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI), into the fusogenic lipid bilayer and treating it to cells in vitro to observe for plasma membrane staining versus endocytic localizations. The targeting and in vivo gene silencing efficacies were previously quantified in a mouse model of Staphylococcus aureus pneumonia, in which the targeting peptide is expected to help the F-pSiNPs to home to the site of infection. Beyond its application in S. aureus infection, the F-pSiNP system may be used to deliver any oligonucleotide for gene therapy of a wide range of diseases, including viral infections, cancer, and autoimmune diseases.

We demonstrate the synthesis of fusogenic porous silicon nanoparticles for effective in vitro and in vivo oligonucleotide delivery. Porous silicon nanoparticles are loaded with siRNA to form the core, which is coated by fusogenic lipids through extrusion to form the shell. Targeting moiety functionalization and particle characterization are included.

用于寡核苷酸传递的可生多孔硅纳米粒子的合成、功能化和表征

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic ...

工程学

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

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

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 ...

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

聚电解质复合微晶体的组装与特性

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

化学

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

用于细胞质 siRNA 传递的带电反应性聚合物纳米粒子的制备

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

长期沉默的1S Intersectin的在小鼠肺的特异性siRNA，通过阳离子脂质体重复交货。通过电子显微镜的击倒效果评价

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

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

摘要

探索更多视频

此视频中的章节

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

多孔硅微粒进行交付的siRNA治疗的

结合QD - FRET技术和微流体实时监测DNA纳米复合自组装

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

用于寡核苷酸传递的可生多孔硅纳米粒子的合成、功能化和表征

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

聚电解质复合微晶体的组装与特性

用于细胞质 siRNA 传递的带电反应性聚合物纳米粒子的制备

长期沉默的1S Intersectin的在小鼠肺的特异性siRNA，通过阳离子脂质体重复交货。通过电子显微镜的击倒效果评价

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

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

摘要

探索更多视频

此视频中的章节

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

多孔硅微粒进行交付的siRNA治疗的

结合QD - FRET技术和微流体实时监测DN​​A纳米复合自组装

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

用于寡核苷酸传递的可生多孔硅纳米粒子的合成、功能化和表征

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

聚电解质复合微晶体的组装与特性

用于细胞质 siRNA 传递的带电反应性聚合物纳米粒子的制备

长期沉默的1S Intersectin的在小鼠肺的特异性siRNA，通过阳离子脂质体重复交货。通过电子显微镜的击倒效果评价

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

结合QD - FRET技术和微流体实时监测DNA纳米复合自组装