Name: testing the in vitro and in vivo efficiency of mrna-lipid nanoparticles formulated by microfluidic mixing
Uploaded: 2023-01-20T13:20:00
Description: Watch this Scientific Journal Video about Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing at JoVE.com

M.J.M. 感谢美国国立卫生研究院 （NIH） 主任新创新者奖 （DP2 TR002776）、科学界面 （CASI） 的 Burroughs Wellcome 基金职业奖、美国国家科学基金会职业奖 （CBET-2145491） 以及美国国立卫生研究院（NCI R01 CA241661、NCI R37 CA244911 和 NIDDK R01 DK123049）的额外资助。

注意：在配制 mRNA-LNP 时，始终保持无 RNase 条件，方法是用 RNase 和 DNA 的表面去污剂擦拭表面和设备。仅使用不含 RNase 的吸头和试剂。
所有动物程序均按照宾夕法尼亚大学的《实验动物护理和使用指南》和宾夕法尼亚大学机构动物护理和使用委员会（IACUC）批准的协议进行。
1.制剂前准备
<ol><li>用 200-300 mL 新鲜的 10x 磷酸盐缓冲盐水 （PBS） ...

<ol>
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通过该工作流程，可以配制和测试各种mRNA-LNP的体外和体内效率。可电离脂质和赋形剂可以以不同的摩尔比和不同的可电离脂质与mRNA重量比进行交换和组合，以产生具有不同物理化学性质的mRNA-LNPs22。在这里，我们配制了摩尔比为 35/16/46.5/2.5（可电离脂质：辅助脂质：胆固醇：脂质-PEG）的 C12-200 mRNA-LNP，可电离脂质与 mRNA 重量比为 10：1。这些 LNP 在体外 HepG2 ?...

近年来，脂质纳米颗粒（LNPs）在非病毒基因治疗领域展现出巨大的前景。2018 年，美国食品和药物管理局 （FDA） 批准了有史以来第一个 RNA 干扰 （RNAi） 疗法 Onpattro by Alnylam，用于治疗遗传性转甲状腺素蛋白淀粉样变性 1,2,3,4。这是脂质纳米颗粒和基于RNA的疗法向前迈出的重要一步。最近，Moderna 和辉瑞/BioNTech 的 mRNA-LNP 疫苗获得了 FDA 批准，用于对抗 SARS-CoV-2 4,5。在每一种基于 LNP 的核酸疗法中，LNP 都有助于保护其货物免受核酸酶降解，并促进有效的细胞内递送 6,7。虽然 LNP 在 RNAi 疗法和疫苗应用中取得了成功，但 mRNA-LNP 也被探索用于蛋白质替代疗法8 以及 Cas9 mRNA 的共同递送和引导 RNA 的递送，用于递送用于基因编辑的 CRISPR-Cas9 系统9。然而，没有一种特定的制剂适合所有应用，LNP制剂参数的细微变化会极大地影响体内的效力和生物分布8,10,11。因此，必须开发和评估单个 mRNA-LNP，以确定每种基于 LNP 的疗法的最佳配方。
LNP 通常由四种脂质成分配制而成：可电离脂质、磷脂、胆固醇和脂质锚定聚乙二醇 （PEG） 偶联物 （lipid-PEG）11,12,13。LNP促进的有效细胞内递送部分依赖于可电离脂质成分12。该成分在生理pH值下呈中性，但在内体11的酸性环境中带正电。离子电荷的这种变化被认为是内体逃逸的关键因素12,14,15。除了可电离脂质外，磷脂（辅助脂质）成分还改善了货物的包封并有助于内体逃逸，胆固醇提供稳定性并增强膜融合，脂质-PEG 最大限度地减少循环中 LNP 聚集和调理作用10,11,14,16.为了配制LNP，将这些脂质成分结合在有机相（通常是乙醇）中，并与含有核酸货物的水相混合。LNP配方工艺用途广泛，因为它允许以不同的摩尔比轻松替换和组合不同的组分，以配制许多具有多种物理化学性质的LNP配方10,17。然而，在探索种类繁多的LNP时，至关重要的是，必须使用标准化程序对每种配方进行评估，以准确测量表征和性能的差异。
本文概述了mRNA-LNP的配方及其在细胞和动物中性能评估的完整工作流程。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 M Hydrochloric Acid</td>
			<td>Sigma</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m Syringe Filters</td>
			<td>Genesee</td>
			<td>25-243</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL BD Slip Tip Syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (C14-PEG2000)</td>
			<td>Avanti Polar Lipids</td>
			<td>880150P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)</td>
			<td>Avanti Polar Lipids</td>
			<td>850725P</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf Tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr>
			<td>200 proof Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>23G Needles</td>
			<td>Fisher Scientific</td>
			<td>14-826-6C</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL BD Disposable Syringes with Luer-Lok tips</td>
			<td>Fisher Scientific</td>
			<td>14-823-435</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL Dialysis Cassettes</td>
			<td>Thermo Scientific</td>
			<td>A52976</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Black Wall Black Bottom Plate</td>
			<td>Fisher Scientific</td>
			<td>07-000-135</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well White/Clear Bottom Plate, TC Surface</td>
			<td>Thermo Scientific</td>
			<td>165306</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate, 1 Kilogram</td>
			<td>Research Products International</td>
			<td>&#160;631-61-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Citrate dibasic</td>
			<td>SIgma</td>
			<td>3012-65-5</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Luer-Lok Syringe sterile, single use, 5 mL</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>C12-200 Ionizable Lipid</td>
			<td>Cayman Chemical</td>
			<td>36699</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 Mice</td>
			<td>Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>57-88-5</td>
			<td></td>
		</tr>
		<tr>
			<td>CleanCap FLuc mRNA (5moU)</td>
			<td>TriLink Biotechnologies</td>
			<td>L-7202</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable cuvettes</td>
			<td>Fisher Scientific</td>
			<td>14955129</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin, Potassium Salt</td>
			<td>Thermo Scientific</td>
			<td>L2916</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>Thermofisher Scientific</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Exel Insulin Syringes - 0.5 mL</td>
			<td>Fisher Scientific</td>
			<td>1484132</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Hep G2 [HEPG2]</td>
			<td>ATCC</td>
			<td>HB-8065</td>
			<td></td>
		</tr>
		<tr>
			<td>HyPure Molecular Biology Grade Water</td>
			<td>Cytiva</td>
			<td>SH30538.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Infinite 200 PRO Plate Reader</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>Perkin Elmer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Kimwipes</td>
			<td>Fisher Scientific</td>
			<td>06-666-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferase Assay Kit</td>
			<td>Promega</td>
			<td>E4550</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoAssemblr Ignite Cartridges - Classic - 100 Pack</td>
			<td>Precision Nanosystems</td>
			<td>NIN0065</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoAssemblr Ignite Instrument</td>
			<td>Precision Nanosystems</td>
			<td>NIN0001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS - Phosphate-Buffered Saline (10x) pH 7.4, RNase-free</td>
			<td>Thermo Scientific</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermofisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>QB Citrate Buffer, (Citrate 100 mM) pH 3.0</td>
			<td>Teknova</td>
			<td>Q2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-it RiboGreen RNA Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>R11490</td>
			<td></td>
		</tr>
		<tr>
			<td>Reporter Lysis 5x Buffer</td>
			<td>Promega</td>
			<td>E3971</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Away Surface Decontaminant</td>
			<td>Thermofisher Scientific</td>
			<td>7000TS1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate</td>
			<td>Sigma</td>
			<td>7601-54-9</td>
			<td></td>
		</tr>
		<tr>
			<td>TNS reagent (6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt)</td>
			<td>Sigma</td>
			<td>T9792</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>9036-19-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Panalytical</td>
			<td>NanoZS</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing the in vitro and in vivo efficiency of mrna-lipid nanoparticles formulated by microfluidic mixing

最近，随着 Moderna 和辉瑞/BioNTech 成功开发 COVID-19 mRNA 疫苗，脂质纳米颗粒 （LNP） 引起了广泛关注。这些疫苗证明了mRNA-LNP疗法的疗效，并为未来的临床应用打开了大门。在 mRNA-LNP 系统中，LNP 作为递送平台，保护 mRNA 货物免受核酸酶降解并介导其细胞内递送。LNP 通常由四种组分组成：可电离脂质、磷脂、胆固醇和脂质锚定聚乙二醇 （PEG） 偶联物 （lipid-PEG）。在这里，通过微流控混合含有LNP脂质成分的有机相和含有mRNA的水相来制备封装编码萤火虫荧光素酶的mRNA的LNP。然后使用基于生物发光板的测定法在体外测试这些 mRNA-LNP，以评估它们在 HepG2 细胞中的转染效率。此外，在通过侧尾静脉静脉注射后，在C57BL / 6小鼠中在体内评估mRNA-LNP。全身生物发光成像是通过使用体内成像系统进行的。显示了 mRNA-LNP 特性、它们在 HepG2 细胞中的转染效率以及 C57BL/6 小鼠中的总发光通量的代表性结果。

mRNA-LNPs采用微流控仪器配制，其平均流体动力学直径为76.16 nm，多分散指数为0.098。通过进行 TNS 测定，发现 mRNA-LNP 的 pKa 为 5.7518。使用改良荧光测定法和 方程式 4.4 计算出这些 mRNA-LNP 的包封效率为 92.3%。用于细胞处理和动物给药的总RNA浓度为40.24 ng/μL。该值是从改良的荧光测定19中获得的，特别是通过将用1%Triton X-100和1x TE缓冲液稀?...

Watch this Scientific Journal Video about Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing at JoVE.com

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

在这里，提出了一种用于配制脂质纳米颗粒（LNP）的方案，该脂质纳米颗粒封装编码萤火虫荧光素酶的mRNA。这些 LNP 在体外 HepG2 细胞和体内 C57BL/6 小鼠中测试 了它们的效力。

测试通过微流控混合配制的mRNA-脂质纳米颗粒的 体外 和 体内 效率

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and ...

该方案可用于配制包裹MRA的一致脂质纳米颗粒。可以调整配方参数，并可以评估各种脂质纳米颗粒的效力和生物分布。脂质纳米颗粒配方以及体外和体内测试的标准化方案可用于配制可以相互比较的一致脂质纳米颗粒。在制定和评估纳米颗粒效力时，有很多小细节需要牢记，但哪些细节考虑在内？制备制剂应该不困难。首先用 200 至 300 毫升新鲜的 10X PBS 填充干净的 4 升烧杯，然后用超纯水将其稀释至 1X。确保 PBS 的最终体积在 2 到 3 升之间。将适当容量的脂质纳米颗粒或LNP制剂的透析盒放入现场烧杯中。使用超纯水将 100 毫摩尔柠檬酸缓冲原液稀释 10 倍，以制备用于 mRNA 稀释的 10 毫摩尔柠檬酸缓冲液。接下来，通过将每种脂质溶解在 100% 乙醇中，制备 C 12 200 可电离脂质、辅助脂质、胆固醇和脂质锚定的 PEG 或脂质 PEG 储备溶液。通过在 37 摄氏度下加热和混合，确保储备溶液完全溶解。将锥形管中不同脂质成分的计算体积组合在一起。用100%乙醇稀释脂质至最终体积为V，其代表LNP最终体积的25%。根据可电离脂质与mRNA的重量比和mRNA-LNP的总体积计算制剂所需的mRNA量。接下来，在冰上解冻编码mRNA的萤火虫荧光素酶。然后使用 10 毫摩尔柠檬酸缓冲液将 mRNA 稀释至有机相的最终体积三倍。用表面去污剂喷洒 RNA 和 DNA 的精细擦拭布，并彻底擦拭微流体仪器的内部。接下来，打开一个新的微流体滤芯，并在入口通道朝下并背对的情况下插入它。在支架上放置两个无菌的 15 毫升锥形管，盖上盖子。完成后，用含有在 10 毫摩尔柠檬酸缓冲液中稀释的 mRNA 的 mRNA 溶液填充 5 毫升注射器。然后用含有在100%乙醇中稀释的脂质的脂质溶液填充三毫升注射器。确保从注射器中去除任何空气。然后不带针头，将含有mRNA溶液的5毫升注射器插入墨盒的左侧入口，将含有脂质溶液的三毫升注射器插入墨盒的右侧入口。打开微流控仪器，选择快速运行。在为插入的 5 毫升和 3 毫升注射器选择正确的注射器类型后，以 3 比 1 的水与有机流速比输入 LNP 配制参数，然后按下开始按钮配制 mRNA-LNP。使用注射器将收集在右锥形管中的mRNA-LNP装入先前水合的透析盒中，而不刺穿或接触透析盒膜，将含有mRNA-LNP的透析盒放回含有PBS的烧杯中，并让它们透析至少两个小时。透析后，将含有mRNA-LNP的透析盒放入无菌生物安全柜中，并使用带针的注射器取出mRNA-LNP。使用0.22微米注射器过滤器无菌过滤mRNA-LNP，并将其收集在无菌锥形管中。然后将 65 微升 mRNA-LNP 溶液放入 1.5 毫升微管中进行表征。将 5， 000 个 HepG2 细胞置于白壁、透明底部、96 孔板中，置于 100 微升完全细胞培养基中，并将细胞在含有 5% 二氧化碳的 37 摄氏度受控培养箱中孵育过夜。在用mRNA-LNP处理细胞之前，从孔中取出完全培养基。接下来，在孵育 24 小时之前，用 100 微升在完全细胞培养基中以所需剂量稀释的 mRNA-LNP 或 100 微升完全培养基处理细胞。孵育后，从 96 孔板中取出细胞培养基，向每个孔中加入 20 微升裂解缓冲液，然后加入 100 微升荧光素酶测定试剂。保护板免受光线照射，并将板放入酶标仪中以测量生物发光信号。使用无菌PBS稀释mRNA-LNP溶液，使100微升尾静脉注射体积中存在2微克的总mRNA。用 100 微升 mRNA-LNP 或 100 微升 PBS 填充 29 号胰岛素注射器，并从注射器中去除任何气泡。将针头斜面朝上插入小鼠的侧尾静脉，并缓慢注射100微升mRNA-LNP或PBS。注射后6小时，在无菌PBS中制备每毫升15毫克的D-荧光素钾盐储备溶液。在活体成像系统（IVIS）的成像软件中，选择发光旁边的框，然后按初始化以准备仪器进行数据采集。接下来，腹膜内给予先前治疗的小鼠200微升D-荧光素，并等待10分钟，同时荧光素酶信号稳定，然后再麻醉小鼠。将麻醉的小鼠转移到体内成像系统内的鼻锥上，确保它们在关闭腔室门之前仰卧，腹部暴露在外。在成像软件中，确保选择了正确的视野并将曝光时间设置为自动，单击软件中的获取按钮即可获得生物发光图像。mRNA-LNP的包封效率为92.3%，使用改良的荧光测定法和方程式。用增加剂量的 mRNA-LNP 处理 5， 000 个 HepG2 细胞时观察到生物发光增加。荧光素酶表达在这项研究的最低剂量（每孔 5 纳克）下很容易检测到。在用配制的LNPs处理的小鼠的上腹部观察到强烈的生物发光信号。 在肝脏信号周围绘制感兴趣的区域以计算总发光通量。总发光通量约为 5 乘以 10 到 9。配制LNP时，始终保持可电离脂质与核酸的重量比。每体积的有机相应含有与三体积水相中的mRNA相对应的可电离脂质。

Research

JoVE Journal

Bioengineering

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Viral Nanoparticles for In vivo Tumor Imaging

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科研

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Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

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Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

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Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

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

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眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

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Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

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液体中脉冲激光烧蚀制备金属纳米粒子：研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

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Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

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Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

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For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

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