ALC-0159

ionizable cationic lipid; RNA delivery

可电离脂质纳米粒中mRNA合成和包封的方案：[1]
基本方案1：通过体外转录和酶盖和拖尾合成mRNA
基本方案2：将mRNA封装到可电离的脂质纳米颗粒中
替代方案：使用预制囊泡对mRNA进行小规模封装
基本方案3：mRNA可电离脂质纳米颗粒的表征和质量控制
更多详细制备方案和实验操作信息，请参阅https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.898

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通过空间机制，ALC-0159 的聚乙二醇 (PEG) 部分有助于纳米颗粒的稳定性 [1]。
Pfizer-BioNTech新冠肺炎疫苗中的LNP含有低水平（＜2 mol%）的ALC-0159，这通过其聚（乙二醇）（PEG）部分的立体机制有助于纳米粒子的稳定。在Moderna新冠肺炎疫苗中，ALC-0159被另一种聚乙二醇化脂质（1,2-二肉豆蔻酰rac-甘油-3-甲氧基PEG2000）取代。根据早期报道的静脉注射聚乙二醇化纳米药物的一些受者的过敏反应，人们猜测ALC-0159（聚乙二醇化脂质）可能在引发过敏反应中发挥作用。[1]

mRNA疫苗最近因其在新冠肺炎大流行期间的成功而引起了人们的极大兴趣。他们的成功是由于mRNA设计和封装到可电离脂质纳米颗粒（iLNP）中的进步。这突出了mRNA-iLNPs在其他环境中的应用潜力，如癌症、基因治疗或不同传染病的疫苗。在这里，我们描述了使用适合用作疫苗和治疗剂的市售试剂生产mRNA iLNP。本文详细介绍了通过酶盖和拖尾的体外转录合成mRNA，以及使用可电离脂质DLin-MC3-DMA将mRNA封装到iLNP中的方案。DLin-MC3-DMA经常被用作新制剂的基准，并为筛选mRNA设计提供了一种有效的递送载体。该方案还描述了制剂如何适应其他脂质。最后，提出了一种用于mRNA iLNP表征和质量控制的逐步方法，包括测量mRNA浓度和包封效率、粒径和ζ电位。[2]

[1]. Allergic Reactions and Anaphylaxis to LNP-Based COVID-19 Vaccines. Mol Ther. 2021 Mar 3;29(3):898-900.

ALC-0315 is an ionisable aminolipid that is responsible for mRNA compaction and aids mRNA cellular delivery and its cytoplasmic release through suspected endosomal destabilization. The LNPs in the Pfizer-BioNTech COVID-19 vaccine contain low levels (<2 mol %) of ALC-0159, which contributes to nanoparticle stabilization by a steric mechanism through its poly(ethylene glycol) (PEG) moiety. Considering their low ALC-0159 content, LNPs in the Pfizer-BioNTech COVID-19 vaccine most likely display a weak steric barrier of PEG. [1]

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Refer to Figure 1 for safe stopping points indicated by red arrows.
Basic Protocol 1: Synthesis of mRNA by in vitro transcription and enzymatic capping and tailing
Allow 2 to 4 days to complete the entire protocol including the production and assessment of capped and tailed mRNA. Four days will be required if precipitations are planned overnight. The IVT, capping and tailing reactions all take approximately half a day. All reactions can be set up in ∼1 hr followed by the required incubation time of two hours for IVT and one hour for capping/tailing. Precipitation of the mRNA requires a 30 min centrifugation step and resuspension of the RNA takes ∼10 min. Quality assessment of the mRNA by Nanodrop, agarose gel and automated gel electrophoresis takes 1 hr.
Note when scaling up, more time is required particularly when multiple or larger mRNA pellets need to be resuspended in nuclease-free water.

Basic Protocol 2: Encapsulation of mRNA into iLNPs
Preparation of lipid solutions may be carried out in advance of the formulation step. Otherwise, the entire encapsulation protocol must be carried out on the same day. Allow an hour for all the reagents to come to room temperature before use and an hour to carry out the formulation and dilution into DPBS step. The centrifugal concentration step is dependent on the particle size, total sample volume and desired end volume. Typically allow 1 to 4 hr. Once concentrated, the iLNP solution can be stored in the fridge until the dilution requirements are determined by the RiboGreen assay.
Alternate Protocol: Small-scale encapsulation of mRNA using preformed vesicles
Same as for Basic Protocol 2.

Basic Protocol 3: Characterization and quality control of mRNA iLNPs
Allow 1 to 2 hr for the RiboGreen assay including allowing the kit to warm to room temperature from the fridge. DLS (size, PDI, zeta potential) should be carried out on the final, diluted sample before it is used for biological evaluation. Preparation of samples takes a few minutes and analysis time varies across different instruments, but it is typically 5 to 10 min per sample.
The buffers used in the TNS assay must be at room temperature before use. Depending on the size of the aliquot this may take several hours. Buffers can be moved to the fridge the night before the assay is to be run to reduce the time required to warm the buffers. The assay requires 40 aliquots in a 96-well plate per iLNP sample therefore allow 10 to 15 min per iLNP sample to prepare the plate and 10 min to read the plate.

Allow 30 min for the mRNA extraction protocol. See above (Basic Protocol 1) for analysis of extracted mRNA by automated gel electrophoresis.[3]

C33H70N2O3

542.9205

542.538

1849616-42-7

155977658

White to off-white solid

39.8Ų

1

4

31

38

415

0

BPWFJNQUTKVHIR-UHFFFAOYSA-N

InChI=1S/C33H67NO3/c1-4-6-8-10-12-14-16-18-20-22-24-26-28-34(33(35)32-37-31-30-36-3)29-27-25-23-21-19-17-15-13-11-9-7-5-2/h4-32H2,1-3H3

2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide

ALC0159; ALC 0159; ALC-0159; Azane;2-(2-methoxyethoxy)-N,N-di(tetradecyl)acetamide; mPEG-DTA; PEG-N,N-ditetradecylacetamide; ALC-0159

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL
Ethanol :≥ 50 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (Infinity mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (Infinity mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (Infinity mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。