Name: formulating and characterizing lipid nanoparticles for gene delivery using a microfluidic mixing platform
Uploaded: 2021-02-25T13:00:00
Description: Watch this Scientific Journal Video about Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform at JoVE.com

感谢阿图尔·萨卢贾、亚廷·戈卡恩、玛丽亚-特雷莎·佩拉基亚、沃尔特·施文格和菲利普·扎卡斯对LNP发展的指导和贡献。

图1提供了整个过程的示意图。
1. 准备缓冲器
注意：这里强烈建议对缓冲器进行无菌过滤，以去除可能影响核酸和 LNP 质量的任何颗粒物。
<ol><li>磷酸盐缓冲盐水 （PBS）<ol>
<li>使用 8 mM Na2HPO 4、2 mM KH2PO4、137 mM NaCl 和 2.7 mM KCl 在核酸酶无水中准备 1x PBS，并将 pH 值调整为 7.4。</li>
		<li>使用 0.22 μm 孔?...

<ol>
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与其他现有方法（如脂膜水化和乙醇注射）相比，可重复性、速度和低体积筛选是使用微流体混合形成 LNP 的重要优势。我们已经证明了这种方法的可重复性，对用不同的LNP批次观察到的封装效率或颗粒大小没有影响。这是任何治疗，包括LNPs，成为临床可用的基本标准。
这里描述的技术采用交错的鱼骨微流体混合，这导致LNP形成的时间尺度只有几分钟。这种混合使用混沌的?...

药物携带者用于保护和提供具有典型有利特性的治疗，包括低细胞毒性，增加生物可用性，并改善稳定性1，2，3。聚合物纳米粒子、鼠类和脂质粒子以前曾被探索过核酸封装和输送4、5、6、7。脂质已用于不同类型的纳米载体系统，包括脂质体和脂质纳米粒子，因为它们与高稳定性8生物相容。LNPs可以很容易地封装核酸的基因传递9，10。它们保护核酸在系统循环过程中不受血清蛋白酶的降解，并可以改善对特定部位的输送，因为LNPs的表面地形和物理特性会影响其生物分布12。LNPs还改善组织渗透和细胞吸收9。先前的研究表明，在LNP13中，西RNA封装取得了成功，包括首个含有西RNA治疗遗传性转乙酰激素介质淀粉样体病的LNP治疗药物，该疗法于2018年获得美国食品和药物管理局（FDA）和欧洲药品管理局的批准。最近，正在研究LNPs的交付更大的核酸，即mRNA和DNA9。截至2018年，有+22个基于脂质的核酸输送系统正在进行临床试验14。此外，含有LNPs的mRNA目前是主要候选者，并已受雇于COVID-19疫苗15，16。这些非病毒基因疗法的潜在成功需要形成小（+100 nm），稳定和均匀的粒子与高核酸封装。
在LNP配方中使用电离子脂作为主要成分，在复合、封装和递送效率14方面显示出优势。离子脂质通常具有酸分离常数 （pKa） < 7：例如，二烯酰-4-二甲基丁基丁酸酯（D-Lin-MC3-DMA），在FDA批准的LNP配方中使用的电离子脂质，pKa为6.4417。在低pH值下，电离子脂质上的胺组变得质子化和正电荷，允许在mRNA和DNA上与负电荷磷酸盐组组装。胺、"N"、组与磷酸盐、"P"的比例，用于优化组件。N/P比率取决于使用的脂质和核酸，根据配方18而异。形成后，pH值可调整为中性或生理pH值，以便进行治疗管理。在这些 pH 值下，电离脂质也脱质，从而向 LNP 提供中性表面电荷。
电离子脂也有助于内分泌逃生19，20。LNPs在细胞吸收过程中接受内分泌，必须从内皮体中释放出来，以便将mRNA货物送入细胞质或DNA货物到细胞核21。内皮体内部通常比细胞外介质更酸性，使电离子脂质正电荷22，23。带正电荷的电离子脂质可与内分泌脂膜上的负电荷相互作用，从而导致内皮体不稳定，从而释放LNP和核酸。目前正在研究不同的电离子脂质，以提高LNP分布的功效，以及内分泌逃逸14。
LNP 的其他典型成分包括辅助脂质，如磷脂酰胆碱 （PC） 或磷乙醇胺 （PE） 脂质。1，2-二甲苯-sn-甘油-3-磷乙醇胺（多普），1，2-二乙酰-sn-甘油-3-磷 磷胆碱 （DSPC）， 和 1，2 二醇 - sn-甘油-3 磷胆碱 （DOPC） 是常用的辅助脂质24，25.DOPE已被证明形成一个倒置的六边形II（HII）相，并通过膜融合26增强透射，而DSPC已被认为稳定LNPs与圆柱形几何27。胆固醇也被纳入配方，以增加膜刚度，随后有助于LNP的稳定。最后，脂质结合聚乙烯乙二醇（PEG）被纳入配方，以提供必要的石碑屏障，以帮助在粒子自组装27。PEG 还通过防止聚合来提高 LNP 的存储稳定性。此外，PEG 经常被用作隐形组件，可以增加 LNP 的循环时间。然而，这一属性也可能构成挑战，招募LNPs肝细胞通过内源性定位机制驱动的阿波利波蛋白E（ApoE）28。因此，研究已经调查了从LNP扩散PEG的乙酰链长度，发现短长度（C8-14）与LNP分离，更适合ApoE招募相比，更长的乙酰长度28。此外，PEG 与之结合的脂质尾巴饱和程度已证明会影响 LNPs29的组织分布。最近，Tween 20，这是生物药物制剂中常用的表面活性剂，具有长期不饱和脂质的尾巴，与PEG-DSPE相比，在排出淋巴结方面具有很高的转化性，PEG-DSPE主要在注射部位29处对肌肉进行透射。此参数可以优化，以实现所需的 LNP 生物分布。
传统的LNP形成方法包括薄膜水化法和乙醇注射法27。虽然这些是现成的技术，他们也是劳动密集型的，可能会导致低封装效率，并具有挑战性地扩大27。混合技术的进步使得方法更易放大，同时开发出更均匀的粒子27。这些方法包括T结混合，交错鱼骨混合，微流体流体动力学聚焦27。每种方法都有独特的结构，但都允许快速混合含有核酸的一个液态与含有脂质成分的有机相，从而产生高封装的核酸27。在此协议中，利用通过微流体墨盒进行快速和可控的混合，采用交错的鱼骨混合设计。本协议概述了含有 LNPs 的核酸的制备、组装和定性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (C-14 PEG)</td>
			<td>Avanti Polar Lipids</td>
			<td>880151P</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;l Graduated Filter Tips&#160; (RNase-,DNase-, DNA-free)</td>
			<td>USA Scientific</td>
			<td>1121-3810</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;l Graduated Filter Tips (RNase-,DNase-, DNA-free)</td>
			<td>USA Scientific</td>
			<td>1111-2831</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;l Beveled Filter Tips (RNase-,DNase-, DNA-free)</td>
			<td>USA Scientific</td>
			<td>1120-1810</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;l Graudated Filter Tips (RNase-,DNase-, DNA-free)</td>
			<td>USA Scientific</td>
			<td>1120-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>3&#946;-Hydroxy-5-cholestene, 5-Cholesten-3&#946;-ol (Cholesterol)</td>
			<td>Sigma-Aldrich</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile Syringes (1 ml syringe)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-823-434</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile Syringes (3 ml syringe)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-823-436</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer General Use Syringe Needles (BD Blunt Fill Needle 18G)</td>
			<td>Thermo Fisher Scientific</td>
			<td>23-021-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop Centrifuge</td>
			<td>Beckman coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Black 96 well plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-245-177</td>
			<td></td>
		</tr>
		<tr>
			<td>BrandTech BRAND BIO-CERT RNase-, DNase-, DNA-free microcentrifuge tubes (1.5mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-380-813</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Fisher Scientific</td>
			<td>02-002-611</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 500ml Vacuum Filter/Storage Bottle System, 0.22 um pore</td>
			<td>Corning</td>
			<td>430769</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable folded capillary cells</td>
			<td>Malvern</td>
			<td>DTS1070</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol, Pure 200 proof</td>
			<td>Sigma-Aldrich</td>
			<td>459844</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher Brand Semi-Micro Cuvette</td>
			<td>Thermo Fisher Scientific</td>
			<td>14955127</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Conical Tubes (15 mL) (DNase-RNase-free)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM12500</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliporeSigma Amicon Ultra Centrifugal Filter Units</td>
			<td>Thermo Fisher Scientific</td>
			<td>UFC901024</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoAssemblr Benchtop</td>
			<td>Precision Nanyosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9930</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;P7589</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-iT RiboGreen RNA Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>R11490</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>02-004-036</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate, Dihydrate, granular</td>
			<td>Fisher Scientific</td>
			<td>02-004-056</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax i3x</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer Nano</td>
			<td>Malvern</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

formulating and characterizing lipid nanoparticles for gene delivery using a microfluidic mixing platform

基于脂质的药物载体由于体积小、生物相容性和封装效率高，已用于临床和商业上可用的交付系统。使用脂质纳米粒子（LNPs）封装核酸有利于保护RNA或DNA免受降解，同时促进细胞吸收。LNPs 通常含有多种脂质成分，包括电离子脂、辅助脂质、胆固醇和聚乙烯乙二醇 （PEG） 结合脂质。LNPs 可以很容易地封装核酸，由于电离子脂质的存在，在低 pH 值是阴离子的，并允许与负电压 RNA 或 DNA 的复合。在这里，LNPs是通过封装信使RNA（mRNA）或质粒DNA（pDNA）形成的，使用有机相中的脂质成分和在声相中核酸成分的快速混合。这种混合使用精确的微流体混合平台进行，允许纳米粒子自组装，同时保持层流。水动力学大小和多散射是使用动态光散射 （DLS） 测量的。LNP 上的有效表面电荷通过测量 zeta 电位来确定。封装效率的特点是使用荧光染料来量化包裹的核酸。具有代表性的结果表明，该方法具有可重复性，并且不同的配方和工艺参数对已开发的 LNP 具有影响。

在不同的天开发了多批具有相同脂质配方和6的NPP，以证明该技术的可重复性。第1批和第2批导致大小分布重叠，具有类似的多散（图2A），在两个不同批次之间的大小或封装效率方面没有显著差异（图2B）。每批封装效率高（>98.5%），尺寸与77纳米LNP直径相似。颗粒均匀，第 1 批的平均多分散度指数 （PDI） 为 0.15，第 2 批的平均...

Watch this Scientific Journal Video about Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform at JoVE.com

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

脂质纳米粒子是使用微流体混合平台方法开发的，用于mRNA和DNA封装。

使用微流体混合平台为基因传递制订和定性脂质纳米粒子

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

该协议可用于生产高可重复性、快速性和低体积的核酸封装脂质纳米粒子。可根据临床应用调整配方参数，实现所需的生物分布。微流体墨盒的标准鱼骨设计允许混合过程中快速、精确和可控的层流。该方法可以扩展并生成均匀、高度封装的粒子。由于大多数商业认可的基因治疗产品都依赖于病毒方法，因此此程序可以提供基因传递的见解，因为它的非病毒方法允许重复剂量的应用是必要的。首先，将每个液体库存溶液的适当数量添加到具有间歇性涡流的玻璃瓶中。如表所示，随后在最终卷的533微升中加入200种证明乙醇。然后加入适量的mRNA库存，用柠檬酸盐缓冲剂稀释，以达到1.5毫升的最终体积在所需的浓度。要调入微流体通道，请首先将原始参数输入表中概述的仪器软件中。接下来，打开仪表盖，将微流体墨盒放入旋转块中。将至少 500 微升乙醇装入 1 毫升注射器中，注意注射器尖端没有气泡或气隙，并将注射器插入墨盒的右入口。装载一个5毫升注射器与1.5毫升的柠檬酸盐缓冲器，注意没有气泡或缝隙，并插入注射器到墨盒的左入口。将两个 15 毫升圆锥形管放入夹架中，作为废容器，然后单击 Go"混合解决方案。当仪器停止按底部蓝光关闭指示启动时，打开后期并正确处理锥形管和注射器。对于脂质纳米粒子的形成，设置表中指示的适当配方参数，并加载一个 1 毫升注射器，并配有以前准备的脂质混合物。去除注射器尖端的任何气隙或气泡，并将注射器插入墨盒的右侧。将先前准备好的核酸溶液加载到 3 毫升注射器中，注意注射器尖端没有气泡或空气间隙，并将注射器插入墨盒的左入口。将标有样品名称的 15 毫升无 RNAase 圆锥形管放入左管夹中，并将 15 毫升废圆锥体放在右管夹中。关闭仪表盖并单击 Go.脂质和 mRNA 溶液将流经微流体墨盒，脂质纳米粒子溶液将收集在圆锥管中。在制剂过程结束时保留锥形管，然后用 5 毫升 PBS 和生物安全柜稀释脂质纳米粒子。要进行缓冲交换，首先用 2 毫升 PBS、离心机预洗 100 千达顿孔大小的超中心过滤器，然后从底部隔间清空 PBS。将稀释的脂质纳米粒子添加到预洗超中心过滤器的顶部隔间中，进行三次离心机清洗，丢弃流经，并在前两次洗涤后将 5 毫升 PBS 添加到超中心滤芯中。最后一次清洗后，将脂质纳米粒子溶液对照超中心过滤器的墙壁进行几次，以尽量减少纳米粒子的损失，然后再将纳米粒子溶液转移到无核素瓶中。然后添加PBS，使纳米粒子悬浮达到适当的实验浓度和体积。为了评估脂质纳米粒子的封装效率，首先准备PBS中工作核酸溶液的两倍连续稀释，以生成标准曲线，从每毫升500毫微克开始，至少稀释5次。接下来，准备PBS中的纳米粒子样品稀释，以达到围绕标准曲线中点的适当理论浓度。然后准备核糖核糖核酸绿色RNA试剂，通过混合适量的RNA试剂、Triton X100 和 PBS 来检测脂质纳米颗粒内外是否存在RNA。然后，要检测液体纳米粒子外是否存在RNA，只需混合适量的RNA试剂和PBS。将核酸标准和脂质纳米粒子溶液的负载复制到具有黑色荧光能力的 96 井板中，然后添加等量的 RNA 定量试剂，并且没有 Triton X100 到标准和样品复制。在不受光线保护的室温下摇动板读卡器五分钟，以获得样品的彻底混合，然后以 480 纳米的激发波长和 520 纳米的发射波长测量微板读卡器上的荧光。为了测量脂质纳米粒子的水动力学大小和多分散性，首先在PBS中稀释纳米粒子溶液40次，并将溶液添加到半微晶圆。将 cuvette 加载到齐塔西器上，并选择一个标准操作程序来设置仪器，根据纳米粒子的材料、分散剂、温度和细胞类型来测量纳米粒子。然后单击"开始"测量脂质纳米粒子的水动力大小和多分散性。为了测量脂质纳米粒子的 Zeta 潜力，在无核酸易感水中稀释纳米粒子溶液 40 次，并将溶液加载到填充线的 cuvette 中。将 cuvette 加载到 Zeta 电位分析仪中，注意电极与仪器接触，并设置仪器根据脂质纳米粒子的特定组成测量 Zeta 电位。然后单击"开始"测量 Zeta 脂质纳米粒子的潜力。在这项分析中，在不同的天开发了多批与脂质配方相同的脂质纳米粒子和胺与磷酸盐的比例，以证明该技术的可重复性。如所述，第一批和第二批表现出具有类似多分散性的重叠大小分布，在批次之间的大小或封装效率方面没有显著差异。通常，配方参数的变化会诱发一些小而具有统计学意义的差异。例如，将胺与磷酸盐的比例降低会导致封装效率降低 4%，同时纳米粒子的水动力直径也随之增加。脂质纳米粒子配制的电油脂不同，但胺与磷酸盐的比例相同，在封装效率方面有显著变化，颗粒直径略有差异。与封装脂质纳米粒子的mRA相比，质粒DNA的封装导致更大的颗粒，尽管这两种类型的纳米粒子都表现出类似的封装效率。然而，流速过程参数的变化不会影响脂质纳米粒子的发育。脂质纳米粒子应用良好，最近批准的COVID-19疫苗是最佳例子。该技术作为一个伟大的工具集，并通过允许下肢配方筛选为未来的应用铺平道路。

Research

JoVE Journal

Bioengineering

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

科研

生物工程

High-throughput Protein Expression Generator Using a Microfluidic Platform

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

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 ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

由微型条纹分析技术在微流控设备混合不均匀

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...