多孔硅微粒进行交付的siRNA治疗的
Summary
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。
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of the Material/Equipment | Company/Institution | Catalog Number | Comments/Description |
| Polyethylenimine (PEI), branched | Sigma-Aldrich | 408727 | Average molecular weight ~25,000 Da |
| L-Arginine | Sigma-Aldrich | A5006 | Reagent grade, ≥98% |
| Boc-Asp-OH | Sigma-Aldrich | 408-468 | 99% |
| (3-Aminopropyl)triethoxysilane | Sigma-Aldrich | 440140 | 99% |
| Hydrogen peroxide solution | Sigma-Aldrich | 216763 | 30 wt. % in H2O |
| Sulfuric acid | Sigma-Aldrich | 339741 | 100.00% |
| Isopropyl alcohol | Sigma-Aldrich | W292907 | ≥99.7% |
| Ethanol | Sigma-Aldrich | 459844 | ≥99.5% |
| N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) | Sigma-Aldrich | 03449 | ≥99% |
| Albumin from bovine serum | Sigma-Aldrich | A7030-10G | Blocking agent |
| Ataxia-telangiectasia mutated siRNA | Sigma-Aldrich | Designed in-house | |
| Tris Acetate-EDTA buffer | Sigma-Aldrich | T9650 | For DNA agarose gel electrophoresis |
| Penicillin-Streptomycin | Sigma-Aldrich | P4333 | |
| Fetal Bovine Serum | Sigma-Aldrich | F2442 | |
| TWEEN 20 | Sigma-Aldrich | P1379 | Polyethylene glycol sorbitan monolaurate |
| 2-Mercaptoethanol | Sigma Aldrich | M6250 | For Western blot |
| Sodium dodecyl sulfate | Sigma-Aldrich | L3771 | |
| Sodium phosphate monobasic | Sigma-Aldrich | 71496 | For making phosphate buffer |
| Sodium phosphate dibasic | Sigma-Aldrich | 71640 | For making phosphate buffer |
| Anti-Mouse IgG | Sigma-Aldrich | A4416 | Secondary antibody (anti-mouse) for Western blot |
| N-Hydroxysuccinimide (NHS) | Sigma-Aldrich | 130672 | 98% |
| CELLSTAR 96W Microplate Tissue Culture Treated Clear w/ Lid | Greiner Bio-One | 655182 | 96-well plate |
| 10X Tris-Glycine Liquid | Li-Cor | 928-40010 | Transfer buffer for Western blot |
| Paraformaldehyde solution 4% in PBS | Santa Cruz | Sc-281692 | Fixation of cells |
| CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS) | Promega | G5421 | Proliferation assay |
| Phosphate buffered saline | Fisher Scientific | BP399-500 | 10 X Solution |
| Corning cellgro Dulbecco's Modification of Eagle's (Mod.) | Fisher Scientific | MT-15-017-CM | Cell culture media, 1X solution |
| Triton X-100 | Fisher Scientific | AC21568-2500 | Octyl phenol ethoxylate, permeabilization agent |
| Cover glass | Fisher Scientific | 12-530C | |
| Methanol | Fisher Scientific | A412-1 | For Western blot transfer buffer |
| Plastic Cuvettes | Fisher Scientific | 14-377-010 | For size measurements using Zetasizer Nano ZS |
| Molecular BioProducts RNase AWAY Surface Decontaminant | Fisher Scientific | 14-754-34 | Spray for removing RNAse contamination |
| Agarose | Fisher Scientific | BP165-25 | Low melting point, for running RNA samples |
| ProLong Gold Antifade Reagent with DAPI | Invitrogen | P36935 | Antifade reagent with DAPI, nucelus detection |
| Alexa Fluor 488 Phalloidin | Invitrogen | A12379 | Dissolve 300 units in 1.5 ml methanol, detection of filamentous actin |
| SYBR Safe DNA Gel Stain | Invitrogen | S33102 | Visualization of RNA |
| Negative Control siRNA | Qiagen | 1022076 | Control siRNA |
| AllStars Neg. siRNA AF 555 | Qiagen | 1027294 | Fluorescent control siRNA |
| Cell scraper | Celltreat | 229310 | |
| BioLite Multidishes and Microwell Plates | Thermo Scientific | 130184 | 6-well plate |
| Pierce LDS Sample Loading Buffer (4X) | Thermo Scientific | 84788 | Sample loading buffer for Western blot |
| Pierce BCS Protein Assay Kit | Thermo Scientific | 23227 | Protein quantification assay |
| Halt Protease Inhibitor Single-Use Cocktails (100X) | Thermo Scientific | 78430 | Protease inhibitor cocktail, use at 1X |
| M-PER Mammalian Protein Extraction Reagent | Thermo Scientific | 78501 | Protein extraction reagent |
| Sorvall Legend Micro 21R | Thermo Scientific | 75002440 | Centrifuge |
| Beta Actin Antibody | Thermo Scientific | MA1-91399 | β-actin primary antibody (from mouse) for western blor |
| 6X TriTrack DNA Loading Dye | Thermo Scientific | R1161 | DNA loading dye |
| Nuclease-Free Water | Life Technologies | AM9938 | |
| Non-Fat Dry Milk | Lab Scientific | M0841 | For Western blot |
| 2-well BD Falcon culture slides | BD Biosciences | 354102 | 2-well culture slides |
| Amersham ECL Western blot detection reagent. | GE Healthcare Life Sciences | RPN2106 | Western blot detection reagent |
| BA Membranes | GE Healthcare Life Sciences | 10402096 | Nitrocellulose membrane for Wester blot |
| ATM (D2E2) Rabbit mAb | Cell Signaling | 2873S | ATM primary antibody (from rabbit) for Western blot |
| Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling | 7074 | Secondary antibody (anti-rabbit) for Western blot |
| Folded capillary cells | Malvern | DTS 1061 | For zeta potentail measurements using Zetasizer Nano ZS |
| MDA-MB-231 cell line | ATCC | HTB-26 | Mammary Gland/Breast |
| 12% Mini-PROTEAN TGX Gel | Bio-rad | 456-1043 | For Western blot |
| Biorad PowerPac HC | Bio-rad | 164-5052 | Power supply for electrophoresis |
| 10x Tris/Glycine/SDS Buffer | Bio-rad | 161-0732 | Running buffer for Western blot |
| Wide Mini-Sub Cell GT Cell | Bio-rad | 170-4405 | Electrophoresis equipment for DNA agarose gel |
| Mini-PROTEAN Tetra cell | Bio-rad | 165-8000 | Electrophoresis equipment for Western blot |
| ChemiDoc XRS+ System with Image Lab Software | Bio-rad | 170-8265 | Image acquisition and analysis software for gels and blots |
| 4" (10cm) dia., 5x7mm diced Silicon Wafer | Ted Pella | 16007 | Silicon waferfor scanning electron microscopy and atomic force microscopy |
| Thermomixer R | Eppendorf | 22670107 | Shaker |
| Isoton II diluent | Beckman Coulter | 8546719 | Isoton diluent |
| Multisizer 4 Coulter Counter | Beckman Coulter | A63076 | Particle counting analyzer |
| Non-functionalized porous silicon particles | Microelectronics Research Center, University of Texas at Austin | Dicoidal shape. 2.6 μm (diameter) x 0.7 μm (hight), provided in isopropyl alcohol | |
| Zetasizer Nano ZS | Malvern | Particle analyzer system for size and zeta potential | |
| Scanning Electron Microscope | FEI | Particle size and shape | |
| Atomic Force Microscope | Bruker | Particle size and shape | |
| Fluo ViewTM 1000 Confocal Microscope | Olympus | Visualization of fixed and live cells |
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