The authors acknowledge financial support from Houston Methodist Research Institute, the National Natural Science Foundation of China (Nos., 21231007 and 21121061), the Ministry of Education of China (Nos., 20100171110013 and 313058), the National Basic Research Program of China (973 Program No. 2014CB845604), and the Fundamental Research Funds for the Central Universities.

1. PCPS粒子的制备<ol><li>氧化在过氧化氢的30％溶液的非官能化的多孔硅的颗粒在95℃下2小时。在2％的异丙醇（3-氨基丙基）三乙氧基硅烷溶液2天，在65℃缓慢搅拌下胺化的氧化颗粒。 </li><li>离心30分钟，将溶液在18800×g离心并在异丙醇和乙醇三次两次洗涤颗粒，利用短暂超声处理，暂停沉淀。执行步骤1.3和1.4，当离开在乙醇溶液中的颗粒。 </li><li>添加粒子悬浮液（ 例如 ，10微升）的已知体积至10ml保序 ​​稀释剂和计数用颗粒计数仪的粒子，以确定在所述储液颗粒的浓度。 </li><li>激活酸基L-精氨酸（0.1纳摩尔）用N的- （3-二甲氨基丙基） - N&#39;-ethylcarbodiimide盐酸盐（EDC，0.1纳摩尔）/ N </em&gt;羟基琥珀酰亚胺（NHS，0.1纳摩尔）在20ml乙醇2小时，在室温温和搅拌。 </li><li>简要地超声处理粒子原液加1十亿颗粒与L-精氨酸水溶液和离开18小时，在室温缓慢搅拌下进行反应。 </li><li>激活第一天冬氨酸组的N - （叔丁氧羰基）-L-天冬氨酸（BOC-ASP-OH，1纳摩尔）用EDC（0.1纳摩尔）/ NHS（0.1纳摩尔）的20毫升乙醇中进行4小时，在4℃，温和搅拌。 </li><li>溶解50毫克聚乙烯亚胺在10毫升乙醇中，并将该溶液添加到将Boc-ASP-OH的混合物。让该反应进行24小时，在RT下在温和搅拌。 </li><li>激活用EDC（0.1纳摩尔）/ NHS（0.1纳摩尔）的将Boc-ASP-OH / PEI溶液的第二天冬氨酸基，在4℃下进行6小时的与温和搅拌。 </li><li>以获得PCPS颗粒，来自步骤1.5中添加的粒子的溶液注入到步骤1.8将Boc-天冬氨酸-OH / PEI溶液。允许反应进行18小时，在RT下在温和ST<html> irring。 </li><li>离心30分钟，将溶液在18800×g离心并用乙醇洗涤颗粒溶液三次，用短暂的超声处理，暂停沉淀。 </li></ol> 2. PCPS颗粒表征<ol><li>测量用扫描电子显微镜（SEM）的颗粒的尺寸。 <ol><li>将一滴颗粒悬浮液中（10,000颗粒/μl，以乙醇）在一个干净的二氧化硅SEM样品存根和在真空下让干燥的RT。 </li><li>用在透镜检测器测量的SEM图像在8千伏具有3-5毫米的工作距离。 </li></ol></li><li>测量使用粒子分析器系统中颗粒的zeta电位。 <ol><li>混合10微升微粒的悬浮液的（10,000颗粒/μl的在乙醇中）用1ml的10mM磷酸缓冲液（pH7.4）中。 </li><li>将样品加载到毛细管折叠细胞，并根据制造商的说明测量ζ电位。 </li></ol></li></ol>乐"&gt; 3。加载的siRNA成PCPS颗粒<ol><li>干燥PCPS颗粒（从PCPS颗粒制备步骤1.9）在真空下O / N。 </li><li>添加的siRNA（4微克）在无核酸酶水（20微升）到干燥PCPS颗粒和超声简要。使用下面的粒子的siRNA比:2×10 5颗粒/ 0.2微克的siRNA，4×10 5个粒子/ 0.2微克的siRNA，6×10 5个粒子/ 0.2微克的siRNA，8×10 5颗粒/ 0.2微克的siRNA，10× 10 5个粒子/ 0.2微克siRNA和12×10 5个粒子/ 0.2微克的siRNA。 </li><li>孵育3小时，在4℃在振荡器上（1000转），以允许的siRNA，与粒子结合。 </li></ol> 4.优化的siRNA / PCPS粒率<ol><li> DNA样染料添加到20微升的PCPS /对照siRNA的颗粒具有不同的粒子的siRNA比（见装载的siRNA成PCPS颗粒）。 </li><li>加载SAM普莱斯到含有DNA的凝胶染料2％琼脂糖凝胶。 </li><li>在运行缓冲液中进行电泳以120伏的恒定电压进行20分钟。 </li><li>分析与图像采集和分析软件的凝胶。 </li></ol>从PCPS颗粒5.释放的siRNA <ol><li>混合20微升PCPS /对照siRNA的颗粒具有不同的粒子的siRNA比（参见步骤3）在十二烷基硫酸钠（SDS，2％），静置1小时，在室温。 </li><li> DNA样染料添加到样品。 </li><li>负载样品到含有DNA的凝胶染料2％琼脂糖凝胶。 </li><li>在用DNA电泳设备和电源运行缓冲液进行电泳，在120伏的恒定电压进行20分钟。 </li><li>分析与图像采集和分析软件的凝胶。 </li></ol> PCPS颗粒6共聚焦显微镜<ol><li>添加5微升PCPS /荧光对照siRNA的颗粒（10×10 5颗粒/ 0。2微克的siRNA / 20微升）到玻璃盖玻片。 </li><li>激光共聚焦显微镜可视化颗粒层。 </li></ol>精氨酸-PEI /对照siRNA纳米粒子7.表征<ol><li>以降低硅材料并形成精氨酸- PEI /对照siRNA的纳米颗粒，PCPS / siRNA的颗粒（10×10 6 PCPS颗粒/ 2微克的siRNA）加入100微升磷酸盐缓冲盐水和摇晃（1000转），在37℃下2天。 </li><li>离心样品30分钟，在18800×g离心，并收集上清液。 </li><li>测量所形成的精氨酸 - PEI / siRNA的纳米颗粒与动态光散射（DLS）的大小。 <ol><li>混合10微升上清液用1ml 10mM磷酸缓冲液（pH7.4）中，并放入一个塑料杯中。 </li><li>测量使用根据制造商的说明颗粒分析器系统的颗粒的尺寸。 </li></ol></li><li>确定所形成的精氨酸/ PEI-siRNA的大小和形态纳米粒子的原子力显微镜。 <ol><li>取10微升上清和地点的在硅晶片上。 </li><li>可视化用原子力显微镜（AFM）的颗粒。 </li></ol></li></ol> 8.细胞培养<ol><li>培养的MDA-MB-231人乳腺癌细胞中的细胞培养基补充有10％胎牛血清和1％青霉素-链霉素，5％的CO 2，95％湿度和37℃。 </li></ol>活细胞与PCPS颗粒9.共焦显微镜<ol><li>板的细胞在2孔培养滑动在3×10 5个细胞的接种密度/孔24小时。 </li><li>添加装载有荧光对照siRNA（10×10 5颗粒/ 0.2微克粒子的siRNA比，为50nM的siRNA），以细胞PCPS颗粒。 </li><li>拍摄动画的细胞与一个共焦显微镜12小时（有一腔室，5％CO 2，95％湿度和37℃的供给）以下的颗粒博览会肯定。 </li></ol>固定细胞与PCPS颗粒10共聚焦显微镜<ol><li>板的细胞在2孔培养滑动在3×10 5个细胞的接种密度/孔24小时。 </li><li>装有荧光对照siRNA（10×10 5颗粒/ 0.2微克粒子的siRNA比，为50nM的siRNA）PCPS粒子添加到细胞中，并孵育1天，7天和10天。 </li><li>用磷酸盐缓冲盐水洗涤细胞两次，然后修复它们与10分钟的4％多聚甲醛溶液中。 </li><li>用磷酸盐缓冲盐水洗涤细胞。 </li><li>透化的细胞用0.1％辛基酚乙氧基化物10分钟，然后冲洗3次，用磷酸盐缓冲盐水。 </li><li>阻断细胞与来自牛血清（10毫克/毫升）白蛋白在磷酸盐缓冲的盐水在室温10分钟，温和搅拌。 </li><li>为了形象化丝状肌动蛋白，培养细胞与荧光标记的鬼笔（1微升/ 40微升封闭溶液）中20分钟，在RT下在温和搅拌下，然后洗在磷酸盐缓冲盐水。 </li><li>从框架中取出载玻片，并添加抗淬灭剂用4&#39;，6-二脒基-2-苯基吲哚（DAPI）进行可视化的核。 </li><li>加一个玻璃盖在上面，并采取细胞共聚焦显微镜的图像。 </li></ol>细胞PCPS /荧光控制颗粒的siRNA 11.流式细胞仪<ol><li>板的细胞在6孔板中以3×10 5个细胞的接种密度/孔24小时。 </li><li>添加装载有荧光对照siRNA（10×10 5颗粒/ 0.2微克粒子的siRNA比，为50nM的siRNA）向细胞PCPS颗粒孵育24小时。 </li><li>用磷酸盐缓冲盐水洗涤细胞并用细胞刮刀刮下它们。 </li><li>储存的细胞在磷酸盐缓冲盐水与分析前2％胎牛血清。使用未处理的细胞作为阴性对照。 </li><li>执行流程CYtometry。 </li></ol>细胞与PCPS粒子和PCPS /控制粒子的siRNA 12.细胞活性<ol><li>板的细胞在96孔板中以3×10 3个细胞的细胞密度/孔24小时。 </li><li>治疗细胞PCPS颗粒（1.5×10 5 /孔和6×10 5 /孔）或PCPS /对照siRNA的颗粒（10×10 5颗粒/ 0.2微克粒子的siRNA比，为10nM和100nM的siRNA的）48小时和72小时。使用未处理的细胞和细胞用磷酸盐缓冲盐水（体积相同的添加的颗粒）作为对照处理。测定一式三份各样品。 </li><li>根据制造商的说明进行细胞增殖测定。 </li><li>表示的数据作为平均值±标准偏差。 </li></ol>细胞PCPS / ATM突变的siRNA颗粒13印迹<ol><li>板的细胞在6孔板中于细胞密度2×10 5个细胞/瓦特ELL 24小时。 </li><li>孵育细胞PCPS /对照siRNA（50纳米）的颗粒或PCPS / ATM的siRNA（50纳米）的颗粒72小时。使用未处理的细胞作为对照。 </li><li>裂解用蛋白质提取试剂补充有蛋白酶抑制剂混合物的细胞。 </li><li>离心细胞裂解物10分钟，在14000×g离心并回收上清液。 </li><li>用蛋白质定量测定法确定根据制造商的说明中的蛋白质浓度。 </li><li>加样缓冲液（含5微升2-巯基乙醇/ ml的缓冲液）的样品和在99℃下加热它们6分钟。 </li><li>加载的蛋白质样品（20微克/微升）在12％SDS-聚丙烯酰胺凝胶中运行缓冲液，并使用电泳设备和一电源进行聚丙烯酰胺凝胶电泳（1小时，120伏）。 </li><li>使用电泳设备传送凝胶在转移缓冲液（用20％甲醇）到硝酸纤维素膜（1小时，100伏），并的电源。 </li><li>方框用5％干奶的膜1小时。 </li><li>孵育在阻断溶液（来自步骤13.9）在ATM第一抗体（来自兔）的膜在1:1000稀释度O / N。 </li><li>用含有0.1％的聚乙二醇山梨糖醇单月桂酸酯的磷酸盐缓冲盐水洗膜，然后与第二抗体（抗 - 兔）的封闭溶液（来自步骤13.9）孵​​育它以1:2500稀释度1小时。 </li><li>用含有0.1％的聚乙二醇山梨糖醇单月桂酸酯的磷酸盐缓冲盐水洗膜并检测蛋白条带用Western印迹检测试剂使用图像采集和分析软件。 </li><li>用于装载控制，洗膜，重复步骤13.9-13.12使用β肌动蛋白一抗（来自小鼠，1:10,000稀释）和二次抗体（抗小鼠1:4000稀释）。 </li></ol>

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The authors declare that they have no competing financial interests.

这个协议描述为成功递送的siRNA和转染到细胞中的方法。特别是，siRNA的有交付通过使用多功能平台，由聚阳离子官能化的pSi粒子来实现的。使用的siRNA疗法具有很大的潜力， 例如 ，癌症治疗，各种癌基因可以高特异性有针对性。因此，存在一个需求，开发siRNA递送车辆，其可以减轻的siRNA治疗的挑战。总之，我们已经提出了一个协议，表明承诺的安全和高效地交付的siRNA。但是，也有应执?...

小干扰RNA（siRNA）是双链RNA分子，其可以抑制基因的表达。在最近几年，siRNA的已经开发的新一代，显示在临床应用中1-5治疗潜力以供将来使用biodrugs的。然而，成功实施的siRNA治疗仍然是一个相当大的挑战，由于降解核酸酶，细胞内差摄取，转染效率低，并从核内体/溶酶体5低效的释放。许多这些障碍可以通过递送平台的发展，它可以安全，高效地递送的siRNA到患病组织被克服。相比于病毒载体，非病毒平台提供若干优点，如安全，成本低，易于剪裁。特别是，阳 ​​离子纳米粒子，如聚合物和脂质，已证明对于siRNA递送3是有用的。 此前，我们已经开发出一种盘状博士微克递送系统，被称为多级向量（MSV）。这种平台是基于顺序阶段，其中一个车辆从另一个释放。第一阶段是车辆从可生物降解的多孔硅（PSI）制成的微粒，而第二阶段的车辆是纳米颗粒装载有药物或造影剂6,7。的纳米颗粒，其被嵌入在PSI材料，正 ​​在逐渐释放在Si降低8。用Si粒子的一个好处是，在形态和表面特性可以很容易地进行调整，以达到最佳的生物分布和药物释放。最近，成功使用MSV平台用于siRNA的脂质体对肿瘤组织的递送的结果显示在一个卵巢癌和乳腺癌的小鼠模型9，10。 在这项工作中，我们已经制备用于siRNA基于所述MSV平台的原理的通用输送系统。此递送系统的效力先前已被证明我们荷兰国际集团的不同siRNA分子11。该系统是一种聚阳离子官能多孔硅（PCPS）载体，包含的pSi接枝聚乙烯亚胺（PEI）和精氨酸（Arg）的。 PEI可在形成具有siRNA的静电相互作用帮助，而精氨酸和PSI可以起到降低PEI的毒性，如先前demonstarted 11。另外，PEI的存在可以帮助细胞内摄取和内体逃逸，而微粒的pSi使siRNA的保护和持续释放。 PSI的颗粒在生理条件下逐渐降低，由此导致的Arg-PEI / siRNA的纳米颗粒（ 图1），其具有不同的形态，并具有较窄的粒径分布11的形成。对于有关PCPS / siRNA的系统的稳定性的细节，请参考研究由Shen 等人 11。此PCPS平台不同于常规MSV，由于第二阶段的纳米粒子最初不prese核苷酸在载体中，但形成在时间作为第一级托架降解11,12。所述PCPS系统的siRNA的装载效率，细胞毒性和基因沉默效率已在体外进行了评价。使用的siRNA针对共济失调毛细血管扩张症突变（ATM）的原癌基因，其参与DNA修复10转染效率进行了测量。先前，ATM的抑制已经显示降低肿瘤生长的乳腺癌模型10。

<table><tbody><tr><td>Polyethylenimine (PEI), branched</td><td>Sigma-Aldrich</td><td>408727</td><td>Average molecular weight ~25,000&amp;nbsp;Da</td></tr><tr><td>L-Arginine</td><td>Sigma-Aldrich</td><td>A5006</td><td>Reagent grade, &amp;ge;98%</td></tr><tr><td>Boc-Asp-OH</td><td>Sigma-Aldrich</td><td>408-468</td><td>99%</td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich</td><td>216763</td><td>30&amp;nbsp;wt. % in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Sulfuric acid</td><td>Sigma-Aldrich</td><td>339741</td><td>100.00%</td></tr><tr><td>Isopropyl alcohol</td><td>Sigma-Aldrich</td><td>W292907</td><td>&amp;ge;99.7%</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>459844</td><td>&amp;ge;99.5%&amp;nbsp;</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(3-Dimethylaminopropyl)-&lt;em&gt;N&amp;prime;&lt;/em&gt;-ethylcarbodiimide hydrochloride (EDC)</td><td>Sigma-Aldrich</td><td>03449</td><td>&amp;ge;99%</td></tr><tr><td>Albumin from bovine serum</td><td>Sigma-Aldrich</td><td>A7030-10G</td><td>Blocking agent</td></tr><tr><td>Ataxia-telangiectasia mutated siRNA&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td>Designed in-house</td></tr><tr><td>Tris Acetate-EDTA buffer</td><td>Sigma-Aldrich</td><td>T9650</td><td>For DNA agarose gel electrophoresis</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F2442</td><td></td></tr><tr><td>TWEEN 20</td><td>Sigma-Aldrich</td><td>P1379</td><td>Polyethylene glycol sorbitan monolaurate</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma Aldrich</td><td>M6250</td><td>For Western blot</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich</td><td>71496</td><td>For making phosphate buffer&amp;nbsp;</td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>71640</td><td>For making phosphate buffer</td></tr><tr><td>Anti-Mouse IgG</td><td>Sigma-Aldrich</td><td>A4416</td><td>Secondary antibody (anti-mouse) for Western blot</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Hydroxysuccinimide (NHS)</td><td>Sigma-Aldrich</td><td>130672</td><td>98%</td></tr><tr><td>CELLSTAR 96W Microplate Tissue Culture Treated Clear w/ Lid</td><td>Greiner Bio-One</td><td>655182</td><td>96-well plate</td></tr><tr><td>10x Tris-Glycine Liquid</td><td>Li-Cor&amp;nbsp;</td><td>928-40010</td><td>Transfer buffer for Western blot</td></tr><tr><td>Paraformaldehyde solution 4% in PBS&amp;nbsp;</td><td>Santa Cruz</td><td>Sc-281692</td><td>Fixation of cells</td></tr><tr><td>CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS)</td><td>Promega</td><td>G5421</td><td>Proliferation assay</td></tr><tr><td>Phosphate buffered saline</td><td>Fisher Scientific</td><td>BP399-500</td><td>10x Solution</td></tr><tr><td>Corning cellgro Dulbecco&#039;s Modification of Eagle&#039;s (Mod.)</td><td>Fisher Scientific&amp;nbsp;</td><td>MT-15-017-CM</td><td>Cell culture media, 1x solution</td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>AC21568-2500</td><td>Octyl phenol ethoxylate, permeabilization agent</td></tr><tr><td>Cover glass</td><td>Fisher Scientific</td><td>12-530C&amp;nbsp;</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific&amp;nbsp;</td><td>A412-1</td><td>For Western blot transfer buffer</td></tr><tr><td>Plastic Cuvettes</td><td>Fisher Scientific&amp;nbsp;</td><td>14-377-010&amp;nbsp;</td><td>For size measurements using Zetasizer Nano ZS</td></tr><tr><td>Molecular BioProducts RNase AWAY Surface Decontaminant</td><td>Fisher Scientific</td><td>14-754-34</td><td>Spray for removing RNAse contamination</td></tr><tr><td>Agarose</td><td>Fisher Scientific</td><td>BP165-25</td><td>Low melting point, for running RNA samples</td></tr><tr><td>ProLong Gold Antifade Reagent with DAPI&amp;nbsp;</td><td>Invitrogen</td><td>P36935</td><td>Antifade reagent with DAPI, nucelus detection</td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Invitrogen</td><td>A12379</td><td>Dissolve 300 units in 1.5&amp;nbsp;ml methanol, detection of filamentous actin</td></tr><tr><td>SYBR Safe DNA Gel Stain</td><td>Invitrogen</td><td>S33102</td><td>Visualization of RNA</td></tr><tr><td>Negative Control siRNA</td><td>Qiagen</td><td>1022076</td><td>Control siRNA</td></tr><tr><td>AllStars Neg. siRNA AF 555</td><td>Qiagen</td><td>1027294</td><td>Fluorescent control siRNA</td></tr><tr><td>Cell scraper</td><td>Celltreat</td><td>229310</td><td></td></tr><tr><td>BioLite Multidishes and Microwell Plates</td><td>Thermo Scientific</td><td>130184</td><td>6-well plate</td></tr><tr><td>Pierce LDS Sample Loading Buffer (4x)</td><td>Thermo Scientific</td><td>84788</td><td>Sample loading buffer for Western blot</td></tr><tr><td>Pierce BCS Protein Assay Kit</td><td>Thermo Scientific</td><td>23227</td><td>Protein quantification assay</td></tr><tr><td>Halt Protease Inhibitor Single-Use Cocktails (100x)</td><td>Thermo Scientific</td><td>78430</td><td>Protease inhibitor cocktail, use at 1x</td></tr><tr><td>M-PER Mammalian Protein Extraction Reagent</td><td>Thermo Scientific&amp;nbsp;</td><td>78501</td><td>Protein extraction reagent</td></tr><tr><td>Sorvall Legend Micro 21R</td><td>Thermo Scientific</td><td>75002440</td><td>Centrifuge</td></tr><tr><td>Beta Actin Antibody&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>MA1-91399</td><td>&amp;beta;-actin primary antibody (from mouse) for western blot</td></tr><tr><td>6x TriTrack DNA Loading Dye</td><td>Thermo Scientific</td><td>R1161</td><td>DNA loading dye</td></tr><tr><td>Nuclease-Free Water&amp;nbsp;</td><td>Life Technologies</td><td>AM9938</td><td></td></tr><tr><td>Non-Fat Dry Milk</td><td>Lab Scientific</td><td>M0841</td><td>For Western blot</td></tr><tr><td>2-well BD Falcon culture slides&amp;nbsp;</td><td>BD Biosciences</td><td>354102</td><td>2-well culture slides</td></tr><tr><td>Amersham ECL Western blot detection reagent</td><td>GE Healthcare Life Sciences</td><td>RPN2106</td><td>Western blot detection reagent</td></tr><tr><td>BA Membranes</td><td>GE Healthcare Life Sciences</td><td>10402096</td><td>Nitrocellulose membrane for Wester blot</td></tr><tr><td>ATM (D2E2) Rabbit mAb</td><td>Cell Signaling</td><td>2873S</td><td>ATM primary antibody (from rabbit) for Western blot</td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody</td><td>Cell Signaling&amp;nbsp;</td><td>7074</td><td>Secondary antibody (anti-rabbit) for Western blot&amp;nbsp;</td></tr><tr><td>Folded capillary cells</td><td>Malvern&amp;nbsp;</td><td>DTS 1061</td><td>For zeta potentail measurements using Zetasizer Nano ZS</td></tr><tr><td>MDA-MB-231 cell line</td><td>ATCC</td><td>HTB-26</td><td>Mammary Gland/Breast</td></tr><tr><td>12% Mini-PROTEAN TGX Gel</td><td>Bio-rad</td><td>456-1043&amp;nbsp;</td><td>For Western blot</td></tr><tr><td>Biorad PowerPac HC</td><td>Bio-rad</td><td>164-5052</td><td>Power supply for electrophoresis</td></tr><tr><td>10x Tris/Glycine/SDS Buffer</td><td>Bio-rad</td><td>161-0732</td><td>Running buffer for Western blot</td></tr><tr><td>Wide Mini-Sub Cell GT Cell</td><td>Bio-rad</td><td>170-4405</td><td>Electrophoresis equipment for DNA agarose gel&amp;nbsp;</td></tr><tr><td>Mini-PROTEAN Tetra cell</td><td>Bio-rad</td><td>165-8000</td><td>Electrophoresis equipment for Western blot</td></tr><tr><td>ChemiDoc XRS+ System with Image Lab Software</td><td>Bio-rad</td><td>170-8265</td><td>Image acquisition and analysis software for gels and blots</td></tr><tr><td>4&quot; (10&amp;nbsp;cm) dia., 5&amp;nbsp;x&amp;nbsp;7&amp;nbsp;mm diced Silicon Wafer</td><td>Ted Pella&amp;nbsp;</td><td>16007</td><td>Silicon waferfor scanning electron microscopy and atomic force microscopy</td></tr><tr><td>Thermomixer R</td><td>Eppendorf</td><td>22670107</td><td>Shaker&amp;nbsp;</td></tr><tr><td>Isoton II diluent</td><td>Beckman Coulter</td><td>8546719</td><td>Isoton diluent</td></tr><tr><td>Multisizer 4 Coulter Counter</td><td>Beckman Coulter</td><td>A63076</td><td>Particle counting analyzer</td></tr><tr><td>Non-functionalized porous silicon particles&amp;nbsp;</td><td>Microelectronics Research Center, University of Texas at Austin</td><td></td><td>Dicoidal shape. 2.6 &amp;mu;m (diameter) x 0.7 &amp;mu;m (height), provided in isopropyl alcohol</td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern</td><td></td><td>Particle analyzer system for size and zeta potential&amp;nbsp;</td></tr><tr><td>Scanning Electron Microscope</td><td>FEI</td><td></td><td>Particle size and shape&amp;nbsp;</td></tr><tr><td>Atomic Force Microscope</td><td>Bruker</td><td></td><td>Particle size and shape</td></tr><tr><td>Fluo ViewTM 1000 Confocal Microscope</td><td>Olympus</td><td></td><td>Visualization of fixed and live cells</td></tr></tbody></table>

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.

这个协议描述了使用非病毒递送系统的安全及有效的siRNA转染。 SEM结果显示，该PCPS颗粒是圆柱形的，并有一个直径为2.6微米（ 图2A）。颗粒被带正电荷的具有大约8.21的ζ电位（ 图2B），从而使静电带负电荷的核苷酸结合。所述PCPS颗粒的不同层的共焦图像证实荧光对照siRNA在多孔硅粒子（ 图2C）内加载。从PSI颗粒精氨酸-PEI / siRNA的纳米颗粒的形成和释放被证实...

Watch this Scientific Journal Video about Porous Silicon Microparticles for Delivery of siRNA Therapeutics at JoVE.com

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

递送仍然是治疗性的实施的小干扰RNA（siRNA）的主要挑战。该协议涉及使用多官能的和生物相容siRNA递送平台，其中包括精氨酸和聚乙烯亚胺接枝的多孔硅微粒。

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 ...

porous-silicon-microparticles-for-delivery-of-sirna-therapeutics

Research

JoVE Journal

Bioengineering

10.9K 次观看.  Houston Methodist Research Institute. 递送仍然是治疗性的实施的小干扰RNA（siRNA）的主要挑战。该协议涉及使用多官能的和生物相容siRNA递送平台，其中包括精氨酸和聚乙烯亚胺接枝的多孔硅微粒。 

视频: Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

用于向癌细胞输送 sirna 的外质体的制备

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

科研

癌症研究

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 ...

生物工程

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

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.

使用微流体装置生产siRNA负载的脂质纳米颗粒

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based ...

生物化学

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

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 regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

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 ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

多孔硅薄膜作为神经生长因子载体的设计

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

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 ...