Name: preparation of neutrally-charged, ph-responsive polymeric nanoparticles for cytosolic sirna delivery
Uploaded: 2019-05-02T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery at JoVE.com

作者感谢 Craig Duvall 博士和 Rebecca Cook 博士为开展这项研究提供了数据和实验室资源。作者感谢 Vanderbilt 纳米科学与工程研究所 (VINSE) 获得 DLS 和 TEM (NSF EPS 1004083) 仪器。作者感谢国家科学基金会支持研究生研究奖学金计划 (NSF#1445197)。提交人感谢国家卫生研究院的财政支持 (NIH R01 EB019409)。提交人感谢国防部国会指导医学研究计划提供的财政支持 (DOD CDMRP OR130302)。

1. 西门子的制备和表征
<ol><li>si-NP 制剂<ol>
<li>将聚合物溶解在 10 mM 柠檬酸缓冲液 (pH 4.0) 中 3.33 mg/mL。聚合物可以首先在乙醇中的10倍浓度下溶解, 以确保溶解。 注: 聚合物可以在较低的浓度下溶解, 但在浓度高于 3.33 Mg\ ml 的情况下使用可以防止均匀的 NP 形成。</li>
		<li>加入 siRNA (直径为50μm), 使 n +:P-比率为 10.通过移液将聚合物和 siRNA 溶液彻底混合, 孵育30分钟。N+</...

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这里描述的硅-nps 是由阴离子 siRNA 和阳离子聚合物与聚离子复合物 (聚多层) 的静电结合而成的。PEG-DB 聚合物的静态络合促进了 Peg-db 聚合物的阳离子复合 (4.0)。在 pH 值4.0 时, DMAEMA 具有很高的质子化作用, 因此 DB 块的电脉冲很高。这确保了聚合物作为 unimers 溶解在溶液中, 而不是形成胶束, 并且数据库复合体高效率地与 siRNA。随后, 溶液的 pH 值调整为中性 (pH 7.4), 导致 DB 块的 "疏水化"、胶束的形成...

由于 sirna 抑制了蛋白质从 mrna 序列中的转化, 理论上可以用于药物治疗所有已知的疾病1,2,3,4,5。然而, sirna 在医学中的应用受到 sirna分子 6,7的综合不良药代动力学特性的限制。静脉注射时, sirna 通过肾脏迅速清除, 并被核酸酶8, 9 降解。由于其巨大的体积和负电荷, siRNA 不能进入细胞或逃避内皮溶酶体通路, 以访问存在于细胞溶胶 10,11,12中的 rna 诱导沉默复合体 (risc), 13岁因此, 广泛的努力集中在 siRNA 交付战略的设计和实施14。这项工作主要集中在开发基于脂质和聚合物的纳米粒子, 这些纳米颗粒包裹 sirna, 保护其免受体内清除和降解, 并通过可电离的阳离子胺基团启动细胞吸收和体内逃逸。临床前的许多成功已经报告, 最近, 第一个临床成功已报告纳米颗粒为基础的肝 siRNA 交付治疗遗传性转胸腺素介导 (hATTR) 淀粉样变 15.
目前有许多致癌基因被传统的药理学 (即小分子药物) "不能下药", 这些基因激励着聚合物 siRNA 纳米颗粒 (si-NPs) 的设计来治疗癌症16。然而, 对于非肝 siRNA 的传递, 必须考虑一组单独的设计参数。输送系统必须保护导致全身循环内的阳离子电荷17,18,19。具体而言, 对于肿瘤的传递, si-np 稳定性对于延长循环, 从而通过增强的渗透性和保留率 (epr) 效应20,21增加肿瘤内的积累是必不可少的。此外, 控制 si-NP 尺寸是必不可少的, 因为只有纳米颗粒直径约 20-200 nm 大小利用 EPR22, 和较小的 si-NP (~ 20–50 nm 直径) 表现出更好的肿瘤渗透比较大的纳米粒子和微粒子23。
为了解决这些额外的设计限制, 在静脉给药后的 siRNA 的系统肿瘤交付, 已经开发出了中电性强的、抗 ph 反应的 sinps (图 1)24。这些硅-nps 是聚乙二醇化的, 或最近的, Zwitter电离 25, 用于中性表面电荷和抗蛋白质吸附和在循环中的光氧化。由于他们不能仅仅依靠阳离子性质来驱动细胞内的传递, 非常有效的子宫内膜逃逸是实现强大的基因沉默的必要条件。因此, 这些 si-NPs 的核心是由一个高度内溶酶的核心, 在细胞外 pH 值 (7.4) 时是惰性的, 但在内索染色体通路的酸化条件下, 这种核心是以类似开关的方式触发的 [pH 6.8 (早期内生体)-5.0 (溶酶体)]。最后, 锡素核心中的阳离子和疏水含量的混合物提供静电和范德华稳定力, 与单纯的阳离子系统相比, 提高了血液中西诺的稳定性。
使用可逆加法-碎片链转移 (RAFT) 控制聚合来生产结构复杂、成分精确的聚合物, 可以将许多功能集成到相对简单的设计中。为了生产具有中性表面电荷、ph 响应能力和 NP 稳定性的硅-NP, RAFT 用于合成聚 (乙二醇-b-[2-[二甲基) 乙基甲基丙烯酸甲酯 (peg-db);图 1a)。PEG-DB 与 siRNA 静电络合, 形成具有 PEG 电晕和 Db/sirna 核的 Sip-nps (图 1b)。PEG 在 si-NP 日冕上形成一个惰性的、带电的亲水层。DB 块由50:50 摩尔比 2-(二甲基氨基) 甲基丙烯酸乙酯 (DMAEMA) 和甲基丙烯酸丁酯 (BMA) 组成。阳离子 DMAEMA 静电复合物负电荷 siRNA。BMA 自联内的 np 核心由范德华斯相互作用, 提高 NP 稳定性。DMAEMA 和 BMA 结合在一起, 将 ph 依赖性脂质双裂解行为传递给 db 聚合物块。在细胞外 pH 值时, DB 块被隔离到 si-NP 核, 并对脂质双层体惰性。在酸性条件下, 如在内皮酶体途径内, DB 块内的可电离 DMAEMA 促进了海绵上的原生质效应, 其中子宫内膜缓冲导致渗透肿胀和破裂26。此外, DB 块内的疏水 BMA 摩尔体积极整合和裂解脂质双层, 从而产生有效的内溶。因此, siRNA 与 PEG-DB 相结合, 形成在细胞外 pH 值时具有中子电荷和高度稳定的 sinps, 但在酸性 pH 值时会破坏脂质双层, 从而确保 siRNA 有效载荷的细胞质传递。
本文介绍了从 PEG-DB 中生产锡-Nps 的实验过程。提出并讨论了测定西-np 理化参数和生物活性的方法。为了快速评估 si-NP 的生物活性, 利用荧光素酶作为敲除研究的模型基因。萤火虫荧光素酶是导致萤火虫 "发光" 的蛋白质.因此, 用萤火虫荧光素酶基因转染的哺乳动物细胞产生一种生物发光 "辉光", 可以使用流量计来量化荧光酶表达水平。在这里, 我们使用荧光素酶来评估西 n 的生物活性, 通过传递 siRNA 来对抗荧光素酶, 并量化与接收拼接 sirna 的细胞相比, 荧光素表达细胞中生物发光的相应减少。

<table>
	<tbody>
		<tr>
			<td>0.45 mm pore-size syringe filters</td>
			<td>Thermo Fisher Scientific</td>
			<td>F25133</td>
			<td>17 mm diameter, PTFE membrane</td>
		</tr>
		<tr>
			<td>0-14 pH test strips</td>
			<td>Millipore Sigma</td>
			<td>P4786</td>
			<td></td>
		</tr>
		<tr>
			<td>10x TAE buffer</td>
			<td>Thermo Fisher Scientific/Invitrogen</td>
			<td>AM9869</td>
			<td></td>
		</tr>
		<tr>
			<td>6-7.7 pH test strips</td>
			<td>Millipore Sigma</td>
			<td>P3536</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well black walled plates</td>
			<td>Corning</td>
			<td>3603</td>
			<td>Tissue-culture treated</td>
		</tr>
		<tr>
			<td>Agarose Powder</td>
			<td>Thermo Fisher Scientific/Invitrogen</td>
			<td>16500</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Millipore Sigma</td>
			<td>C1909</td>
			<td></td>
		</tr>
		<tr>
			<td>dibasic sodium phosphate dihydrate</td>
			<td>Millipore Sigma</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr>
			<td>D-luciferin</td>
			<td>Thermo Fisher Scientific</td>
			<td>88294</td>
			<td>Monopotassium Salt</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995065</td>
			<td>High glucose and pyruvate</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Millipore Sigma</td>
			<td>459836</td>
			<td></td>
		</tr>
		<tr>
			<td>ethidium bromide</td>
			<td>Thermo Fisher Scientific/Invitrogen</td>
			<td>15585011</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>loading dye</td>
			<td>Thermo Fisher Scientific/Invitrogen</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferase siRNA</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>Antisense Strand Sequense: GAGGAGUUCAUUAUCAGUGCAAUUGUU Sense Strand Sequense: CAAUUGCACUGAUAAUGAACUCCT*C* *DNA bases</td>
		</tr>
		<tr>
			<td>MDA-MB-231 / Luciferase (Bsd) stable cells</td>
			<td>GenTarget Inc</td>
			<td>SC059-Bsd</td>
			<td>Luciferase-expressing cells sued to assess si-NP bioactivity</td>
		</tr>
		<tr>
			<td>monobasic sodium phosphate monohydrate</td>
			<td>Millipore Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>Scarmbled siRNA</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>Antisense Strand Sequense: AUACGCGUAUUAUACGCGAUUAACGAC Sense Strand Sequense: CGUUAAUCGCGUAUAAUACGCGUA*T* *DNA bases</td>
		</tr>
		<tr>
			<td>square polystyrene cuvettes</td>
			<td>Fisher Scientific</td>
			<td>14-955-129</td>
			<td>4.5 mL capacity</td>
		</tr>
		<tr>
			<td>TEM grids</td>
			<td>Ted Pella, Inc.</td>
			<td>1GC50</td>
			<td>PELCO Center-Marked Grids, 50 mesh, 3.0mm O.D., Copper</td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Millipore Sigma</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>uranyl acetate</td>
			<td>Polysciences, Inc.</td>
			<td>21447-25</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of neutrally-charged, ph-responsive polymeric nanoparticles for cytosolic sirna delivery

SiRNA 作为靶向分子医学的成功取决于它对病理组织内细胞的有效细胞质传递。用 siRNA 治疗以前 "不可吸毒" 的肝病目标的临床成功已经取得。然而, 有效的肿瘤 siRNA 传递需要额外的药代动力学设计考虑, 包括长循环时间, 逃避清除器官 (如肝脏和肾脏), 以及肿瘤的渗透和保留。在这里, 我们描述了聚合物纳米粒子的制备和体外物理化学生物学特性, 设计用于高效 siRNA 传递, 特别是非肝组织, 如肿瘤。SiRNA 纳米颗粒是通过 siRNA 和多聚二块共聚物 (乙二醇-b-[2-(二甲基氨基) 甲基丙烯酸乙酯) (peg-db) (聚乙二醇-db) (聚乙二醇-db) 制备的聚离子复合物 ((即 siRNA 被隔离在多聚核中, PEG 形成亲水性、含中子电晕。此外, DB 阻滞成为膜溶解作为囊泡的内皮溶酶体途径酸化 (< pH 6.8), 触发内染色体逃逸和细胞质传递 siRNA。介绍了分析 siRNA 纳米粒子的大小、表面电荷、粒子形态和 siRNA 负载等物理化学特性的方法。以荧光素酶为模型基因, 测定了 siRNA 纳米粒子的生物活性, 并进行了快速、高通量的基因沉默检测。通过这些初步测试的设计 (如基于 Peg-dbs 的多指) 被认为适合转换为评估 siRNA 输送到肿瘤或其他病理部位的临床前动物研究。

有效的 sinp 在体内 Srna 传递的一些基本特征是适当的大小 (约 20-200 nm 直径)、siRNA 包装和基因沉默的生物活性。虽然这不是一个详尽无遗的清单 (如讨论中所述), 但在考虑进一步测试某一提法之前, 应确认这些基本特征。
图 2说明了在配方时 si-NP 尺寸和表面电荷的特性。DLS 和 TEM 被用作观察 si-NP 尺寸 (两?...

Watch this Scientific Journal Video about Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery at JoVE.com

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

介绍了制备和表征含中子、ph 响应的 siRNA 纳米粒子的物理化学特性和生物活性的方法。讨论了成功的 siRNA 纳米胺的标准, 如大小、形态、表面电荷、siRNA 负载和基因沉默。

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 ...

目前的协议是重要的，因为它允许研究人员生产siRNA加载的纳米粒子，并充分描述这些纳米粒子，以确保其适合给给动物之前，随后的研究。这种方法的优点是生产纳米粒子，提高siRNA的生物利用度，并产生中性充电的纳米粒子，从而适用于全身管理。这项技术是有影响的，因为siRNA纳米粒子可以应用于治疗无数需要全身给普的疾病，包括癌症、心血管疾病和自身免疫性疾病等。首先，以每毫升33.3毫克的浓度将聚合物溶解在乙醇中，搅拌以确保溶解。然后使用移液器将聚合物溶液转移到10毫摩尔柠檬酸缓冲液中，达到每毫升3.33毫克的浓度。将蒸馏水加入含有小干扰RNA的管中，达到50微摩尔的浓度。通过上下移液将聚合物和小型干扰RNA溶液彻底混合到管中，实现氮磷比10。将管子放在环境温度下 30 分钟。然后将 pH 8 下 10 毫升磷酸盐缓冲液加入管中，达到每毫升 0.28 毫克的浓度，然后通过移液或倒置管轻轻混合。为了确认最终pH值是中性的，大约7.2至7.5，移液器10微升的小干扰RNA纳米粒子溶液进入pH测试条。首先，通过0.45微米孔径注射器过滤器将每毫升0.28毫克浓度的小干扰RNA纳米颗粒过滤成方形石英或聚苯乙烯，制备动态光散射样品。然后根据制造商的规格使用动态光散射仪器记录尺寸和表面电荷测量。为了确认使用传输电子显微镜的小干扰RNA纳米粒子的大小和形态，首先在每个TEM网格中添加5微升过滤小干扰RNA纳米粒子溶液，每毫升0.28毫克。将网格放在环境温度下 60 秒。用滤纸擦干三秒钟。接下来，在每个网格中加入5个3%的醋酸兰基溶液微升，孵育20秒。干干三秒钟。然后将网格放在干燥器中干燥过夜，然后根据传输电子显微镜进行成像。要执行气糖凝胶迟钝，首先在烧杯中 pH8 的 pH 8 中加入两克电泳级 agarose 粉末至 100 毫升 1X Tris-醋酸-EDTA 缓冲液，以产生 2%的 agarose 凝胶。搅拌暂停阿加罗斯。将烧杯盖在微波炉中加热一到三分钟，直到所有气糖溶解。冷却后，以每毫升10毫克浓度加入5微升溴化乙酰胺，并混合好。然后将糖倒入凝胶托盘中，放置梳子以产生井。让凝胶干燥30分钟。小心地拆下梳子，留下装料井，将 1X 三丝醋酸盐-EDTA 缓冲液倒入凝胶托盘中，以填充到最大填充线。然后，在石蜡膜上放置两个微升的负载染料等同物，每个小干扰RNA纳米粒子配方的含量量为不同的氮磷比。移液器将10微升的小干扰RNA纳米颗粒溶液与石蜡膜上的加载染料混合。将混合物加入阿加罗斯凝胶井。将电泳系统插入电源，以 100 伏电压源运行 35 分钟，或直到样品穿过 80% 的凝胶长度。将凝胶从托盘转移到紫外线透光器，并根据制造商的规格可视化紫外透光器上的小干扰RNA波段。根据凝胶底部小干扰RNA波段的消失，确定最佳氮磷酸比。为了击倒模型基因荧光素酶，首先在96井的黑色壁板中播种荧光素酶表达细胞，DMEM在10%FBS中，密度为每井2000个细胞。将盘子放入培养箱过夜，让细胞粘附。早晨去除介质，每井添加10微升小干扰RNA纳米颗粒稀释到全血清介质中，最终小干扰RNA浓度为100纳米摩尔。将孔返回培养箱24小时，用小干扰RNA纳米粒子处理细胞。第二天，用含有每毫升150微克的完全血清介质更换介质。在室温下孵育细胞五分钟，然后测量板读卡器上的发光度。然后重复介质茶点两次，使用不含D-西西法林的新鲜全血清介质，然后孵育24小时。用含有每毫升D-荧光素150毫克的全血清介质更换介质，并在室温下孵育5分钟。测量 48 小时时间点的亮度。对于纵向研究，在生物安全柜外将盖子放在井盘顶部，以在无菌条件下维护细胞，并在用新鲜全介质替换含荧光素的介质后继续孵育。动态光散射测量表明，小干扰RNA纳米粒子一的平均直径为35纳米。峰值宽度较窄的单峰的存在表示单模分布和低多分散度。TEM 测量确认动态光散射的大小测量表明存在一致的小干扰RNA纳米粒子，并揭示了小干扰RNA纳米粒子的球形形态。制备步骤中聚合物浓度过高，导致小干扰RNA纳米粒子2个直径超过200纳米，多模态和多分散体，溶液中的聚合体形成没有明显的颗粒形态。两个小干扰RNA纳米粒子一和二显示中性表面电荷，以接近零的均值zeta电位值表示。Agarose凝胶迟钝测定表明，凝胶底部小干扰RNA波段的消失，表明小干扰RNA与聚合物的复合。聚合物复合小干扰RNA无法通过凝胶迁移，因此在装料井附近的凝胶顶部被可视化。随着氮与磷酸盐比的增加，小干扰RNA复合增加，如凝胶底部小干扰RNA带强度降低所示。与搅乱控制相比，生物活性荧光素酶小干扰RNA纳米粒子一个表现出显著减少的荧光素酶活性。相比之下，荧光石小干扰RNA纳米粒子二不被认为是生物活性的。为了产生具有所需物理化学特性的一致siRNA纳米粒子，应使用具有适当浓度的聚合物溶液，并在协议的每一步验证缓冲溶液的pH值。利用这些方法在我们的研究中，我们已经能够生产基于siRNA纳米粒子的疗法，用于以前无法治疗的致癌基因，并测试这些疗法在乳腺癌临床前模型中的有效性。

Research

JoVE Journal

Bioengineering

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

制定通过Nanoprecipitation技术嵌段聚合物纳米粒子

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

科研

生物工程

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

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

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

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

编程干细胞的治疗性血管新生使用可生物降解高分子纳米微球

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Harmonic Nanoparticles for Regenerative Research

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

多孔硅微粒进行交付的siRNA治疗的

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

Polymeric Microneedle Array Fabrication by Photolithography

聚合物微针阵列制造用光刻

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

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

制造超疏水高分子材料生物医学应用

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

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

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

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