Name: polyethyleneimine-coated iron oxide nanoparticles as a vehicle for the delivery of small interfering rna to macrophages in vitro and in vivo
Uploaded: 2019-02-05T13:00:00
Description: Watch this Scientific Journal Video about Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo at JoVE.com

这项工作得到了国家自然科学基金 (81772308) 和国家重点研究开发计划 (编号: 2017yfa0205502) 的支持。

所有涉及活体动物的方法都是按照中国东南大学的动物护理和使用指南进行的。
1. pei-spions 的制备
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油酸修饰 spions 的制备
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<li>在 n2 的保护下, 在水中溶解 fecl 3·6h2o 和 feso 4·7h2o.<ol>
<li>在烧杯中加入28克 fecl 3·6h2o 和20克 feso4·7h2o, 加入80毫升的去离子水中.通过玻璃导管将 ...

<ol>
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巨噬细胞很难通过常用的非病毒方法进行转染, 如电穿孔、阳离子脂质体和脂质种。在这里, 我们描述了一个可靠和有效的方法来转染巨噬细胞与 sirna。使用目前的协议, 90% 以上的巨噬细胞样 raw 264.7 细胞 (图 2b) 和大鼠腹腔巨噬细胞18可以用 sirna 转染, 而不会对细胞的存活能力造成重大损害。该方法依赖于由氧化铁芯和 pei 壳组成的纳米载体--的输送平台 pei-s...

巨噬细胞是一种分布在所有身体组织中的先天免疫细胞, 尽管数量不同。通过产生多种细胞因子和其他介质, 它们在宿主防御入侵微生物病原体、损伤后组织修复和维持组织稳态1方面发挥着至关重要的作用。巨噬细胞由于其重要性, 一直是深入研究的主题。然而, 尽管 sirna 介导的基因沉默在基因调控和功能研究中很普遍, 但它在巨噬细胞中成功的可能性较小, 因为这些细胞--特别是初级巨噬细胞--往往难以转染。这可归因于与最成熟的转染方法相关的毒性相对较高, 在这种方法中, 细胞膜是化学的 (例如, 与聚合物和脂质的) 或物理的 (例如, 通过电穿孔和基因枪) 被破坏, 让 sirna 分子穿过膜, 从而极大地降低了巨噬细胞的生存能力2,3。此外, 巨噬细胞是富含降解酶的专用吞噬细胞。这些酶会损害 sirna 的完整性, 削弱其沉默效率, 即使基因特异性 sirna 已被传递到细胞3,4。因此, 一个有效的巨噬细胞靶向 sirna 传递系统需要在传递过程中保护 sirna 的完整性和稳定性4。
越来越明显的是, 功能失调的巨噬细胞与某些常见临床疾病 (如自身免疫性疾病、动脉粥样硬化和癌症) 的发生和发展有关。因此, 调节巨噬细胞功能, 例如, sirna, 已经成为一种有吸引力的方法来治疗这些疾病5, 6,7.虽然已经取得了很大进展, 但基于 sirna 的治疗策略的一个主要挑战是系统管理的 sirna 的细胞特异性差, 巨噬细胞的 sirna 吸收不足, 从而导致不受欢迎的副作用。与游离核酸疗法相比, 由于其自发倾向于被网状内皮系统捕获, 通常缺乏最佳的细胞选择性, 并经常导致离靶不良效应, 药物负载纳米粒子 (nps),可设计为在体内被动靶向巨噬细胞, 允许提高治疗效果与最小的副作用8。目前探索的用于传递 rna 分子的 np 包括无机纳米载体、各种脂质体和聚合物9。其中, 聚乙基烯胺 (pei) 是一种能够将核酸结合并凝结成稳定的 np 的阳离子聚合物, 它显示出最高的 rna 传递能力9,10。pei 保护核酸免受酶和非酶降解, 介导它们在细胞膜上的转移, 并促进它们在细胞内的释放。虽然最初作为 dna 传递试剂引入, pei 后来被证明是一个有吸引力的平台, 在本地或系统 9,10体内 sirna 传递。
超顺磁性氧化铁纳米粒子 (spions) 由于其磁性能、生物相容性、与生物重要物体的可比尺寸、较高的表面积与体积比以及易于适应的特性, 在生物医学领域显示出巨大的前景表面活性剂附件11的表面。例如, 由于 spions 作为造影剂的潜在效用和巨噬细胞的快速吸收, 已成为一种最受欢迎的临床工具, 可对巨噬细胞进行成像。虽然 spions 也被广泛研究为核酸交付车辆11,13,14, 15,据我们所知, 文献中很少有关于 spions 作为载体的报告。巨噬细胞靶向 sirna 的传递。对于 spions 的基因传递, 它们的表面通常涂有一层亲水阳离子聚合物, 负电荷核酸可以被静电吸引和捆绑在上面。在这里, 我们提出了一种方法, 合成 spions 的表面被修改与低分子量 (10 kda), 分枝 pei (pei-spions)。然后利用这些磁性纳米平台浓缩 sirna, 形成 pei-spion/sirna 复合物, 使 sirna 能够进入细胞。我们认为, 网状内皮系统16细胞自发吞噬的 spions, 再加上 pei 结合和凝结核酸的强大能力, 使得 pei-spions 适合有效地将 sirna 运进 sirna巨 噬 细胞。这里提供的数据支持了 pei-spion/sina 介导的基因沉默在培养和体内巨噬细胞中的可行性。

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td>Warm in 37&#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>A31608-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin (1.5 ml)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrazolium-based MTS assay kit</td>
			<td>Promega</td>
			<td>G3582</td>
			<td>For cytotoxicity analysis</td>
		</tr>
		<tr>
			<td>RAW 264.7 cell line</td>
			<td>Cell Bank of Chinese Academy of Sciences, Shanghai, China</td>
			<td>TCM13</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plates (6-well)</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dishes (10 cm)</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free tubes (1.5 ml)</td>
			<td>AXYGEN</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 ml)</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Wistar rats</td>
			<td>Shanghai Experimental Animal Center of Chinese 
			Academy of Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacillus Calmette&#8211;Gu&#233;rin freeze-dried powder</td>
			<td>National 
			Institutes for Food and Drug Control, China</td>
			<td></td>
			<td>for inducing adjuvant arthritis in rats</td>
		</tr>
		<tr>
			<td>siRNA</td>
			<td>GenePharma (Shanghai, China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-siRNA</td>
			<td>RiboBio (Guangzhou, China)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethyleneimine (10 kDa)</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>E107079</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia water</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>A112077</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>O108484</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>D103272</td>
			<td></td>
		</tr>
		<tr>
			<td>FeSO4&#8226;7H2O</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10012118</td>
			<td></td>
		</tr>
		<tr>
			<td>FeCl3&#8226;6H2O</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10011918</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimercaptosuccinic acid</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>D107254</td>
			<td></td>
		</tr>
		<tr>
			<td>ultrafiltration tube</td>
			<td>Millipore</td>
			<td>UFC910096</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylammonium hydroxide solution</td>
			<td>Aladdin Chemical Reagent Co., Ltd.</td>
			<td>T100882</td>
			<td></td>
		</tr>
		<tr>
			<td>Particle size and zeta potential analyzer</td>
			<td>Malvern, England</td>
			<td>Nano ZS90</td>
			<td></td>
		</tr>
	</tbody>
</table>

polyethyleneimine-coated iron oxide nanoparticles as a vehicle for the delivery of small interfering rna to macrophages in vitro and in vivo

巨噬细胞由于在调节免疫反应方面的关键作用, 一直是深入研究的对象, 在自身免疫性疾病、动脉粥样硬化和癌症等许多疾病中都是一个有希望的治疗目标。rnai 介导的基因沉默是探索和操纵巨噬细胞功能的一种有价值的选择方法;然而, 巨噬细胞与 sirna 的转染通常被认为是技术上具有挑战性的, 目前, 很少有专门用于 sirna 转移到巨噬细胞的方法。在这里, 我们提出了一个协议, 使用聚乙烯烯胺涂层超顺磁性氧化铁纳米粒子 (pei-spions) 作为有针对性地传递 sirna 到巨噬细胞的载体。当 fe: sirna 重量比达到4及以上时, pei-spions 能够结合并完全冷凝 sirna。在体外, 这些纳米粒子可以有效地将 sirna 传递到初级巨噬细胞中, 也可以传递到类似巨噬细胞的 raw 264.7 细胞系中, 而不会在最佳转染剂量下损害细胞活力, 最终, 它们会诱发sirna 介导的靶向基因沉默。除了用于体外sirna 转染外, pei-spion 也是一种很有前途的工具, 可将sirna 传递给体内的巨噬细胞。鉴于其磁性能和基因沉默能力的综合特点, 系统管理的 pei-spion/srna 颗粒不仅可以调节巨噬细胞的功能, 而且可以使巨噬细胞被成像和跟踪。从本质上讲, pei-spions 代表了一个简单、安全和有效的非病毒平台, 用于在体外和体内将 sirna 传递给巨噬细胞。

该协议制备的 pei-spions 的大小和 zeta 电位分别在 29-48 nm (多分散指数: 0.12-0.23) 和 30-48 mv 之间。在4°c 的水中, 它们在4°c 下稳定超过 12个月, 没有明显的聚集。为了评价它们的 sirna 结合能力, pei-spions 在不同的 f:sina 重量比条件下与 sirna 混合。图 1显示, 当 fe: sirna 重量比达到4及以上时, 游离 sirna 的带完全缺失, 这意味着成功的 sirna 与 pei-spions 结合。pei ...

Watch this Scientific Journal Video about Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo at JoVE.com

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

我们描述了一种使用聚乙烯烯胺 (pei) 涂层超顺磁性氧化铁纳米粒子与 sirna 转染巨噬细胞的方法。这些纳米颗粒可以有效地传递 sirna 巨噬细胞在体外和体内和沉默目标基因表达。

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 ...

这项措施可以帮助回答关键问题。在人文领域。这个答案在巨噬细胞在生理端的功能下到病理条件。该技术的主要优点是聚乙烯胺涂层可以有效地在体外，而不影响细胞。在最佳剂量下。该技术的含义扩展到许多慢性炎症性疾病和癌症的治疗和诊断。巨噬细胞在发病机制中起着至关重要的作用。演示程序将是娜佳。我实验室的研究生和王开轩是肖华博士实验室的研究生。准备联盟酸改性超级顺磁氧化铁纳米粒子或SPION首次添加28克法拉斯氯化六水合物。和20克法司自酸补液到烧杯含有80毫升的去维水。并使用玻璃导管将氮气引入溶液中。搅拌，直到固体物质溶解。将反应混合物加热至72摄氏度，每分钟搅拌800度。并在烧杯中加入40毫升28%氨水。五分钟后加入九毫升的联盟酸滴明智。并保持混合物在72摄氏度连续搅拌三个小时。在孵育结束时，将产生的溶液冷却至室温。根据标准协议，通过磁分离沉淀混合物。然后用绝对乙醇洗涤含有沉淀物的SPION三次。并在100毫升的n-十六烷中刺激沉淀物。准备一毛钱卡普托丙烯酸改性SPION的添加800毫克的联盟酸修改SPION的。分散在200毫升的n-hexane和400毫克的甲壳酸分散在200毫升丙酮分散到三颈烧瓶在60摄氏度的水浴。接下来，在烧瓶中加入200微升的三乙胺滴法，每分钟搅拌1000次，回流。黑色沉淀物可以在五小时后通过磁分离获得。然后使用四甲酰氢氧化氢基调整溶液的pHH，使脱毒水中的亲水SPION的均匀分散。除型聚乙烯胺涂层SPION的添加一毛甲酸改性SPION的胶体溶液下降到10千吨聚乙烯胺溶液明智。在500毫升三颈烧瓶的机械搅拌下，每分钟旋转1000次，旋转两个小时。在孵化结束时，将产生的溶液加入到超过滤管中，其分子量可减少100千吨，含量为15毫升。将样品中分，直到剩余溶液达到一毫升体积。将去维水加入溶液中，使体积回到15毫升。和中注入和补水解决方案十倍，刚才证明。然后通过 22 微米过滤器过滤溶液，在 4 摄氏度下储存。为了准备siRNA纳米粒子，首先将3微升新鲜准备好的20微摩尔siRNA溶液添加到5个标有RNA的免费微中心熔融管中。并将聚乙烯素SPION的0、8、1.6、3.2和6.4微克分别添加到标有零、1、2、4和8的管中。与温和移液混合后，在室温下孵育样品30分钟，使聚乙烯胺SPION siRNA复合物形成。并准备一个百分之三的阿加罗斯凝胶与我纯度的阿加罗斯。在孵育结束时，每5微升样品添加一个微升6XDNA加载缓冲液，并仔细混合。然后将所有样品加载到凝胶上。以每厘米五伏运行电泳。直到布罗莫酚蓝色染料迁移三分之二的长度在凝胶上。转染前一天用PBS清洗像RAF264.7细胞培养的小鼠微噬细胞。在37摄氏度的37摄氏度下，用1毫升25%的三丁辛治疗细胞，在5%的二氧化碳培养箱中。当大多数单元格分离时。用五毫升的完整 DMEN 灭活。然后多次对溶液进行点状，以分散任何细胞簇。将细胞悬浮液转移到无菌的15毫升锥形管中进行离心。然后重新悬浮在五毫升新鲜完整的DMEN细胞计数。稀释细胞到4.5倍10至第四细胞每毫升完整的DMEN浓度。并在六井板的每个井中加入两毫升的培养，在细胞培养箱中24小时孵化。第二天，在1.5毫升RNA流微离心管中，用温和的混合，准备适当的聚乙烯胺SPION siRNA纳米粒子的比例。并将适当的聚乙烯胺SPION siRNA纳米颗粒复合物添加到每口已用一毫升新鲜介质取代的井中。准备聚乙烯胺SPION si复合物。在这种情况下，可以使用多个月的聚乙烯胺SPION，从而最大限度地降低其潜在的毒性。然后小心地旋转板，实现纳米粒子在井底的均匀分布。将板返回细胞培养箱，直到随后的细胞更新或基因敲解缺乏分析。聚乙烯胺SPION的Zeta电位为30.5和37毫伏，每毫升铁浓度高达30微克，不表现出明显的细胞毒性。这是大约两个折叠雇用比浓度通常用于细胞转染。然而，聚乙烯矿SPION的48毫伏的泽塔电位是有毒的。即使在检查的最低剂量。根据流式细胞学分析，超过90%的细胞可以以每毫升15微克的铁转染荧光标记聚乙烯胺SPION siRNA复合物。通过粉碎蓝色染色，评估聚乙烯聚乙烯SPION siRNA浓度对细胞内化的影响，可发现每毫升7.5微克铁的可检测染色量最低。但是，由于聚乙烯胺SPION siRNA吸收饱和，每毫升15微克铁的斑点在较高的铁浓度下不会增加。与非特异性siRNA相比，与聚乙烯胺SPION的窝藏特异性siRNA相染的食管巨噬细胞，其焦油mRNA水平显著降低。建议siRNA可以从内分泌囊泡逃入细胞质，到达RNA干扰机制。进一步，在静脉注射单剂量聚乙烯胺SPION siRNA纳米粒子复合物的CD11b阳性和CD3阳性细胞的絮体分析后，显示CD11b表达巨噬细胞比CD3阳性细胞在任何时间点所检查的CD3阳性细胞更有效地吸收纳米粒子。在尝试此过程时，必须记住以不高于 37 毫伏的平均数据电位准备聚乙烯胺 SPION，并在低铁与 siRNA 比率上准备聚乙烯胺 SPION siRNA 复合物。这项技术比较了研究人员在人类疾病（如癌症和癌症）的动物马达中寻找靶心巨噬细胞的治疗潜力的需要。

Research

JoVE Journal

Medicine

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

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

科研

医学

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

通过40W的Elvax缓释给药的大鼠视网膜：对慢性疾病的治疗

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

隔离和使用的牛肠系膜动脉和静脉部分，作为生物测定在小肠测试的Vasoactivity

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...