Name: synthesis, functionalization, and characterization of fusogenic porous silicon nanoparticles for oligonucleotide delivery
Uploaded: 2019-04-16T14:45:00
Description: Watch this Scientific Journal Video about Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery at JoVE.com

这项工作得到了国家卫生研究院通过合同 # R01 AI13231313-01 的支持。

1. 多孔硅纳米粒子 (pSINPs) 的合成
注意: 使用氢氟酸 (HF) 时, 请务必小心。根据安全数据表 (SDS) 遵循所有安全指南, 在烟罩中处理任何含 hf 的化学品, 并佩戴适当的个人防护设备 (PPE; 外部有带丁基手套的双手套, 下面有实验室涂层的丁基围裙下面有安全护目镜)。所有大学和研发实验室在使用前都需要进行有关高频安全的专门培训。未经当地实验室安全协调员事先批准, 请勿尝试?...

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多孔硅纳米粒子的合成如图 5所示。在合成杂源 pSiNPs 的关键步骤是在加载步骤 (步骤 3)。如果机身纳米颗粒聚集后合成 (图 3), 原因可能是: (1) 氯化钙库存不是均匀制备的, 因此步骤3.1.2 必须仔细跟踪或细化;或 (2) Psin:sirn:cicl 2 的比率或三个成分中的一个或多个成分的浓度可能不是最佳的。建议从 Ccl 2 浓度开始重新优化 (例如, 从2m 更改?...

基因治疗调节特异性基因表达, 以获得治疗效果。已经发现并研究了许多基因调控工具, 包括使用寡核苷酸的核糖核酸干扰 (rnai) (例如, 短干扰 rna (sirna)1,2, 微 rna (mirna)3,4)、Dna 质粒5、6、核酸酶 (如锌指、talens)7、8和 crispr/cas9 系统9,10。虽然每个工具的作用机制不同, 但所有工具都必须到达细胞的细胞质或细胞核才能活跃。因此, 虽然这些工具已被证明能诱导在体外调节基因表达方面发挥显著作用, 但体内的功效却存在细胞外和细胞内障碍。由于这些工具是生物来源的, 我们的身体中存在许多酶和清除系统, 有能力降解或去除外来分子11。即使在工具到达目标细胞的情况下, 它们也会出现内吞;一种细胞吸收模式, 将工具封装和捕获在酸性胃状囊泡中, 使工具降解或排出细胞。事实上, 研究表明, 脂质纳米颗粒是通过共核细胞增多内植的, 大约70% 的 sirna 是在吸收的24小时内从细胞中排出的,12, 13。大部分剩余的 sirna 通过溶酶体途径降解, 最终只有1-2% 的 sirna 最初进入细胞, 而纳米颗粒实现内染色体逃逸, 有可能经历 rnai13,14.
我们最近开发了具有由多孔硅纳米颗粒组成的 sirna 负载核心和一个熔融脂壳15的杂生多孔硅纳米颗粒 (F-pSiNPs).与其他常规寡核苷酸输送系统相比, F-sisnps 具有三大优势: (1) 一种能使颗粒绕过内吞并直接在细胞细胞质中提供整个有效载荷的致热脂质涂层 (与1-2% 相比)通过内细胞颗粒 13,14) (图 1);(2) pSiNPs (& gt;20 wt% compared to 1-15 wt% by conventional systems)15, which rapidly degrade in the cytoplasm (once the core particles shed the lipid coating via fusogenic uptake) to release the siRNA; 中的 siRNA 的高质量负载(3) 靶向肽共轭, 选择性归巢于体内所需的细胞类型。
F-pSiNP 系统已证明了显著的基因沉默效能 (& gt;95% in vitro; & gt;80% in vivo) and subsequent therapeutic effect in a fatal mouse model of S. aureus pneumonia;其结果是在之前公布的15。然而, F-pSiNP 系统的复杂结构需要精细的处理和微调优化, 以产生均匀和稳定的纳米粒子。因此, 本工作的目的是提出一个完整的协议, 以及优化策略的合成, 功能化, 并表征 F-pSiNPs, 用于有针对性地传递 Sirna, 以实现强大的基因沉默效果。

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			<td height="21" style="height:21px;width:399px;">1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)</td>
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			<td style="width:512px;">Purity: 48%</td>
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			<td style="width:512px;">Sample program available at: http://sailorgroup.ucsd.edu/sofware/</td>
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			<td height="42" style="height:42px;width:399px;">siRNA</td>
			<td style="width:163px;">Dharmacon</td>
			<td style="width:119px;">Custom order</td>
			<td style="width:512px;">IRF5, sense 5&#8217;-dTdT-CUG CAG AGA AUA ACC CUG A-dTdT-3&#8217; and antisense 5&#8217;-dTdT UCA GGG UUA UUC UCU GCA G dTdT-3&#8217;</td>
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			<td height="21" style="height:21px;width:399px;">Sonicator</td>
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			<td style="width:512px;">sequence CRVLRSGSC; made cyclic by a disulfide bond between the side chains of the two cysteine residues</td>
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			<td>Thermo Fisher Scientific</td>
			<td>10977015</td>
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synthesis, functionalization, and characterization of fusogenic porous silicon nanoparticles for oligonucleotide delivery

随着基因治疗的出现, 开发有效的体内核苷酸-有效载荷运载系统已成为平行的重要手段。具有较高的寡核苷酸承载能力和独特的细胞吸收途径, 避免了内吞, 其具有较高的体内基因沉默效率。F-pSiNPs 的合成是一个多步骤的过程, 包括: (1) 硅孔中寡核苷酸有效载荷的加载和密封;(2) 多孔硅芯周围的致热脂质同时涂装和施胶;(3) 结合靶向肽和洗涤, 去除多余的寡核苷酸、硅碎片和肽。粒子的尺寸均匀性以动态光散射为特征, 其核壳结构可通过透射电子显微镜验证。通过将亲脂性染料 1, 1 '-二辛代基-3, 3 ' 3 ', 3 '-四甲基硫代卡宾素高氯酸盐 (DiI) 加载到热原脂质双层 (DiI) 中, 并将其体外处理到细胞中, 观察质膜染色量与质膜的对比, 从而验证了热原吸收的有效性。内细胞定位。靶向和体内基因沉默的效果以前是在金黄色葡萄球菌肺炎的小鼠模型中量化的, 在该模型中, 靶向肽有望帮助 F-pSiNPs 回家到感染部位。除了在金黄色葡萄球菌感染中的应用外, F-pSiNP 系统还可用于提供任何寡核苷酸用于广泛疾病的基因治疗, 包括病毒感染、癌症和自身免疫性疾病。

成功合成杂生 pSiNPs 应能产生均匀、略显不透明的溶液 (图 3a)。如果不能优化 pSiNPs 的比例和浓度: Sirn:ccol2可能会导致加载时聚集 (图 3b)。当颗粒通过200纳米膜挤出时, DLS 测量的致熔剂 pSiNPs 的平均流体动力直径应为约200纳米, 平均 zeta 电位约为 + 7 mV, 如图 4所示。在靶向肽表面改性后, 总直径应增?...

Watch this Scientific Journal Video about Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery at JoVE.com

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

我们证明了富里多孔硅纳米粒子的合成, 以提高体外和体内寡核苷酸的传递效果。多孔硅纳米颗粒加入 siRNA 形成核心, 通过挤压包覆的杂原脂质形成外壳。包括定位功能化和粒子表征。

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 ...

该协议描述了纳米粒子系统的合成，该系统能够通过高效率地提供siRNA来压制体内的基因。只需交换靶向肽和siRNA有效载荷，富生纳米粒子就可用作需要体内基因调制的疾病的潜在治疗配方。我们之前已经发布了结果，使用熏蒸纳米平台对抗克阳性细菌感染，通过沉默一个基因，编码在巨噬细胞炎症，以终止慢性炎症。在执行此过程之前，在三对一氢氟酸溶液中组装含有硅晶片的特氟隆蚀刻电池。运行方波形的交流电，低电流密度为 50 毫微米/厘米平方 0.6 秒，高电流密度为 400 毫微/厘米平方，重复 500 次循环 0.36 秒。蚀刻完成后，将细胞从电路中取出，然后用注射器小心冲洗出氢氟酸溶液。然后，用乙醇冲洗三次。要从硅晶片中提起多孔层，首先用 10 毫升的 1 至 29 氢氟酸溶液填充蚀刻电池的孔。然后，运行每厘米 3.7 毫%的常量，持续 250 秒。蚀刻完成后，从电路中取出电池。多孔硅层可能有可见的纹波，表明与晶体硅晶片分离。轻轻洗出氢氟酸溶液，然后用乙醇和水冲洗三次。使用移液器尖端，牢固地裂开多孔硅层的周长，以完成分离。使用乙醇，将多孔硅碎片或芯片从特氟隆蚀刻细胞收集到称重船上。然后，将芯片转移到玻璃瓶中进行储存。接下来，用两毫升无RNase水代替含有多孔硅片的玻璃瓶中的乙醇。使用 Parafilm 牢固地盖住并密封小瓶。将玻璃瓶放在声波浴中并悬浮，使多孔硅片的体积完全淹没在水浴表面以下。为防止在声波处理过程中出现重大水损失，请放置一个装满水的体积烧瓶，倒置，使烧瓶的开口接触水浴表面。然后，以 35 千赫的射频功率对多孔硅芯片进行 12 小时的声波化。以 48 瓦的射频功率。声音后，将玻璃瓶放在平坦的表面上一小时，使较大的颗粒在底部沉淀。然后，使用移液器收集上流水液。在玻璃瓶中，加入72.55微升的DMPC溶液、15.16微升的DSPE-PEG溶液和19.63微升的DOTAP溶液，通过移液混合。将小瓶放在带松动盖的烟罩中，使溶剂在一夜之间蒸发。干燥的薄膜将是小瓶底部的一种浑浊、坚硬、凝胶状的物质。接下来，在先前制备的冰式声波浴中放置一个含有150微升多孔硅纳米粒子的微离心管。在15分钟的超声波下，将150微升siRNA和700微升的二摩尔氯化钙轻轻移液到多孔硅纳米颗粒中。从声波器上取出含有一毫升 siRNA 加载的镀钙多孔硅纳米粒子的管子。将宽烧杯装满去维化水。将聚碳酸酯膜和四个过滤器支架漂浮在水面上，浸泡它们。然后，按照制造商的说明组装脂体挤出套件。接下来，用一毫升的siRNA涂层多孔硅纳米颗粒，在玻璃瓶中水合干脂膜。移液器，直到所有脂质从小瓶底部抬起，并观察到多云，均匀溶液。在玻璃瓶中加入一个磁性搅拌棒，将小瓶放在热板上，将颗粒加热至40摄氏度，同时以磁性搅拌溶液20分钟。之后，将空注射器连接到挤出机的一侧。然后，将另一个注射器填充一毫升脂涂层多孔硅纳米颗粒溶液，然后将注射器连接到挤出机的另一侧。开始挤出，缓慢地将活塞推入，将颗粒从一个注射器中推出，穿过聚碳酸酯膜，然后推入另一个注射器。重复20次。然后，将挤压的颗粒收集到微离心管中。在无 RNase 水中准备具有每毫升一毫克肽浓度的肽库存。在含有富原脂涂层多孔硅纳米颗粒的微离心管中，加入100微升的肽，轻轻移液器。在室温下保持管静20分钟。为了去除多余的肽或siRNA和其他辅料，在室温下以5000倍g旋转一小时，用30千托离心过滤器清洗。在相同设置下，用一毫升 PBS 多离心机两次。离心后，以所需的浓度在PBS中重新使用最终肽结合的富质脂涂层多孔硅纳米粒子。这些颗粒可以等分，并在零下80摄氏度下储存至少30天。成功合成富索源多孔硅纳米颗粒应产生一种均匀、略不透明的溶液，可储存30天，并在不引起结构变化的情况下解冻。未能优化比率和浓度，以及重复的冻结解冻循环可能会导致加载时聚合。当粒子被挤出时，由动态光散射测量的富生多孔硅纳米粒子的平均流体动力学直径应约为200纳米，平均泽塔电位约为正7毫伏。使用靶向肽进行表面改性后，总直径应小于230纳米，平均泽塔电位降至负3.4毫伏。融合可以通过用嗜脂氟化物DII标记富原脂质，并使用共生显微镜观察体外定位来证实。当富源多孔硅纳米粒子的脂质将DiI转移到等离子膜并独立于晶基进行局部化时，可以观察到成功的融合。不成功的融合将显示细胞细胞质中的DII定位，并和细胞体共同定位。这种富索原纳米粒子系统现在不仅用于细菌感染的治疗应用，还用于癌症辅助治疗和癌症免疫治疗。此协议可以进一步优化，以加载超过siRNA。富原纳米粒子系统在装载和交付更大的反电有效载荷方面显示出潜力，例如mRNA和CRISPR/Cas9复合物。

Research

JoVE Journal

Engineering

Optical Trapping of Nanoparticles

纳米粒子的光学捕获

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

科研

工程学

Synthesis and Functionalization of Nitrogen-doped Carbon Nanotube Cups with Gold Nanoparticles as Cork Stoppers

黄金纳米粒子作为软木塞氮掺杂碳纳米管杯的合成与功能构造

Nitrogen-doped carbon  nanotubes consist of many cup-shaped graphitic compartments termed as  nitrogen-doped carbon nanotube cups (NCNCs).  These ...

Monolayer Contact Doping of Silicon Surfaces and Nanowires Using Organophosphorus Compounds

Monolayer Contact Doping (MLCD) is a simple method for doping of surfaces and nanostructures1. MLCD results in the formation of highly controlled, ultra ...

Selective Area Modification of Silicon Surface Wettability by Pulsed UV Laser Irradiation in Liquid Environment

硅表面润湿性的选择区域改性脉冲紫外激光照射在液体环境

The wettability of silicon (Si) is one of the important parameters in the technology of surface functionalization of this material and fabrication of ...

Silicon Metal-oxide-semiconductor Quantum Dots for Single-electron Pumping

硅金属 - 氧化物 - 半导体量子点单电子泵

As mass-produced silicon transistors have reached the nano-scale, their behavior and performances are increasingly affected, and often deteriorated, by ...

Synthesis and Functionalization of 3D Nano-graphene Materials: Graphene Aerogels and Graphene Macro Assemblies

合成和3D纳米石墨烯材料的功能化：石墨烯气凝胶和石墨烯宏组件

Efforts to assemble graphene into three-dimensional monolithic structures have been hampered by the high cost and poor processability of graphene. ...

Integration of Light Trapping Silver Nanostructures in Hydrogenated Microcrystalline Silicon Solar Cells by Transfer Printing

灯光诱杀银纳米结构通过转移印花在氢化微晶硅太阳能电池集成

One of the potential applications of metal nanostructures is light trapping in solar cells, where unique optical properties of nanosized metals, commonly ...

In Situ Synthesis of Gold Nanoparticles without Aggregation in the Interlayer Space of Layered Titanate Transparent Films

In Situ Synthesis of Gold Nanoparticles without Aggregation in the Interlayer Space of Layered Titanate Transparent Films

在没有聚集的层状钛酸盐薄膜透明的层间金纳米粒子的原位合成

Combinations of metal oxide semiconductors and gold nanoparticles (AuNPs) have been investigated as new types of materials. The in situ synthesis of AuNPs ...

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

利用磁性纳米粒子丰富和扩大罕见抗原特异性 t 细胞

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

Procedure for the Transfer of Polymer Films Onto Porous Substrates with Minimized Defects

聚合物薄膜在多孔基板上的转移程序,具有最小化的缺陷

The fabrication of devices containing thin film composite membranes necessitates the transfer of these films onto the surfaces of arbitrary support ...