Ionizable cationic lipid; RNA delivery
ALC-0159 does not act by binding to specific biological targets; its function is based on physicochemical mechanisms. As a PEG-lipid, it forms a PEG “brush” layer on the LNP surface through steric hindrance. This layer serves two primary purposes: during preparation and storage, it prevents LNP particle aggregation via steric repulsion; in vivo, it reduces adsorption of serum proteins (i.e., reduces opsonization) to evade recognition and clearance by the mononuclear phagocytic system. Proper PEG chain length and density are critical for achieving these “stealth” properties and prolonging circulation half-life. ALC-0159 is typically used at a low molar ratio (approximately 1.5%) in formulations to balance circulation time with target cell uptake efficiency.

Preparation of Lipid Nanoparticles
This section describes the formulation of lipid nanoparticles (LNPs) based on the composition of the FDA-approved COVID-19 mRNA vaccine BNT162b2. The lipid blend consists of ALC-0315, DSPC, cholesterol, and ALC-0159 at a molar ratio of 46.3 : 9.4 : 42.7 : 1.6, respectively. The weight ratio of RNA to total lipid is 0.05 (wt/wt).
A. Preparing the Lipid Phase
1. Each lipid is dissolved in ethanol to create stock solutions at a concentration of 10 mg/mL. These stocks can be stored at 20 °C for future use.
Note 1: The ionizable lipid is typically a viscous liquid and should be measured by weight rather than by pipetting volume to ensure accuracy.
Note 2: Cholesterol solutions must be kept warm (>37 °C) to remain fluid. Avoid allowing them to cool before use.

2. To prepare 1 mL of lipid mixture (containing 10 mg total lipid), combine the following volumes of each 10 mg/mL stock:
- 560 µL ALC-0315
- 261 µL cholesterol
- 117 µL DSPC
- 62 µL ALC-0159
Mix thoroughly until a clear solution is obtained.
Note 3: The choice and proportions of lipids can be modified, which will affect LNP characteristics such as size, polydispersity, efficacy, and the required amount of mRNA.

B. Preparing the mRNA Phase
1. Prepare an mRNA solution at 166.7 µg/mL in 100 mM sodium acetate buffer (pH 5).
Note 4: The lipid-to-mRNA mass ratio influences encapsulation efficiency. Users may adjust this ratio to develop alternative formulations.

C. Mixing Methods
Three common techniques are used to rapidly combine the two phases: pipette mixing, vortex mixing, and microfluidic mixing. The first two methods may produce LNPs with greater heterogeneity, lower encapsulation efficiency, and higher variability, whereas microfluidic mixing offers better control, reproducibility, and uniformity.
1. Pipette mixing method
1.1. Quickly add 3 mL of the mRNA solution to 1 mL of the lipid mixture (a typical ethanol-to-aqueous buffer volume ratio of 1:3). Immediately pipette the combined solution up and down vigorously for 20–30 seconds.
1.2. Allow the mixture to sit at room temperature for up to 15 minutes.
1.3. After mixing, dialyze the LNP suspension against PBS (pH 7.4) for 2 hours, then sterilize by passing through a 0.2 µm filter and store at 4 °C.

2. Vortex mixing method
1.1. Vortex 3 mL of the mRNA solution at moderate speed. While vortexing, rapidly add 1 mL of the lipid mixture (1:3 ethanol-to-buffer ratio). Continue vortexing for another 20–30 seconds.
1.2. Incubate the resulting dispersion at room temperature for up to 15 minutes.
1.3. Dialyze against PBS (pH 7.4) for 2 hours, filter-sterilize through a 0.2 µm filter, and store at 4 °C.

3. Microfluidic mixing method
1.1. Using a microfluidic device, mix 3 mL of the mRNA solution with 1 mL of the lipid mixture at a total flow rate of 12 mL/min (ethanol-to-buffer volume ratio of 1:3).
Note 5: Operating parameters such as flow rate ratio and total flow rate can be adjusted to fine-tune LNP properties.
1.2. After mixing, dialyze against PBS (pH 7.4) for 2 hours, filter through a 0.2 µm filter, and store at 4 °C.

---

Protocols for mRNA Synthesis and Encapsulation in Ionizable Lipid Nanoparticles: [2]
Basic Protocol 1: Synthesis of mRNA by in vitro transcription and enzymatic capping and tailing
Basic Protocol 2: Encapsulation of mRNA into ionizable lipid nanoparticles
Alternate Protocol: Small-scale encapsulation of mRNA using preformed vesicles
Basic Protocol 3: Characterization and quality control of mRNA ionizable lipid nanoparticles.
For more details, please refer to https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.898

---

By means of a steric mechanism, the polyethylene glycol (PEG) moiety of ALC-0159 aids in the stability of nanoparticles [1].
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. In the Moderna COVID-19 vaccine, ALC-0159 is replaced with another PEGylated lipid (1,2-dimyristoyl-rac-glycero-3-methoxyPEG2000). There are speculations on a possible role for ALC-0159 (the PEGylated lipid) in triggering anaphylaxis, based on earlier reported anaphylactic reactions in some recipients of intravenously infused PEGylated nanomedicines.[1]

---

In vitro, the activity of ALC-0159 is primarily reflected in its contribution to nucleic acid delivery efficiency as an LNP component. Ionizable lipid nanoparticles containing ALC-0159 can efficiently encapsulate mRNA, protect it from RNase degradation, and deliver mRNA into the cytoplasm for protein expression. Studies indicate that the molar ratio of ALC-0159 in LNPs must be precisely controlled: too low a ratio results in insufficient stability and aggregation, while too high a ratio leads to an excessively dense PEG layer that inhibits cellular uptake and endosomal escape, thereby reducing transfection efficiency. The approximately 1.5% ALC-0159 ratio in the Pfizer-BioNTech vaccine LNP formulation has been validated as an optimal balance. Its in vitro function is indirectly evaluated through assessment of LNP particle size, encapsulation efficiency, and reporter gene (e.g., luciferase) expression in cells.

The in vivo activity of ALC-0159 is manifested through its function within LNPs. In animal models, LNP-mRNA vaccines containing ALC-0159 induce robust antigen-specific immune responses. The PEGylated lipid confers “stealth” properties to LNPs in vivo, reducing rapid clearance by liver and spleen macrophages and allowing more nanoparticles to reach target tissues (such as draining lymph nodes at the injection site or muscle tissue). In the Pfizer-BioNTech COVID-19 vaccine, LNPs containing ALC-0159 successfully delivered mRNA encoding the SARS-CoV-2 spike protein to antigen-presenting cells, inducing neutralizing antibodies and T cell immunity. Clinical studies in non-human primates and humans have demonstrated favorable immunogenicity and protective efficacy of this formulation.

mRNA vaccines have recently generated significant interest due to their success during the COVID-19 pandemic. Their success is due to advances in mRNA design and encapsulation into ionizable lipid nanoparticles (iLNPs). This has highlighted the potential for the use of mRNA-iLNPs in other settings such as cancer, gene therapy, or vaccines for different infectious diseases. Here, we describe the production of mRNA-iLNPs using commercially available reagents that are suitable for use as vaccines and therapeutics. This article contains detailed protocols for the synthesis of mRNA by in vitro transcription with enzymatic capping and tailing and the encapsulation of the mRNA into iLNPs using the ionizable lipid DLin-MC3-DMA. DLin-MC3-DMA is often used as a benchmark for new formulations and provides an efficient delivery vehicle for screening mRNA design. The protocol also describes how the formulation can be adapted to other lipids. Finally, a stepwise methodology is presented for the characterization and quality control of mRNA-iLNPs, including measuring mRNA concentration and encapsulation efficiency, particle size, and zeta potential.[2]

---

ALC-0159 is not involved in traditional enzyme or receptor binding assays; its evaluation in cell-free systems focuses on the physicochemical characterization of LNPs. A typical protocol: 1) Dissolve ALC-0159 with other lipids (ionizable lipid, cholesterol, helper phospholipid) in anhydrous ethanol at optimized molar ratios. 2) Rapidly mix the lipid ethanol solution with mRNA-containing citrate buffer (pH 4.0) using a microfluidic device for self-assembly into LNPs. 3) Measure average particle size (target 80-120 nm), polydispersity index (PDI <0.2), and Zeta potential (near neutral at physiological pH) using dynamic light scattering. 4) Determine mRNA encapsulation efficiency using the RiboGreen fluorescent dye method. 5) The PEG chain structure of ALC-0159 can be characterized by time-of-flight high-resolution mass spectrometry, showing a polydisperse molecular weight distribution with a mean of approximately 2500 Da.

In vitro cell assays are primarily used to evaluate the delivery efficiency and cytotoxicity of LNPs containing ALC-0159. The protocol is as follows: 1) Prepare ALC-0159-containing LNP-mRNA using standard microfluidic methods. 2) Seed target cells (e.g., HEK293T, DC2.4 dendritic cells, or HeLa cells) in culture plates (approximately 1×10⁵ cells per well) and culture overnight for adhesion or suspension adaptation. 3) Add LNP-mRNA at various concentrations to the culture medium and incubate for 24-48 hours at 37°C with 5% CO₂. 4) Assess delivery efficiency: if delivering mRNA encoding GFP or luciferase, evaluate fluorescence intensity by flow cytometry or fluorescence microscopy, or quantify by luciferase activity assay. 5) Assess cell viability using CCK-8 or MTT assay to evaluate LNP cytotoxicity. 6) Measure cellular uptake efficiency by flow cytometry (using fluorescently labeled mRNA).

In vivo animal assays are primarily used to evaluate the efficacy, biodistribution, and immunogenicity of LNP-mRNA vaccines or therapeutics containing ALC-0159. Protocol using a mouse model: 1) Randomize 6-8 week old female BALB/c or C57BL/6 mice into treatment and control groups (5-8 mice per group). 2) Administer ALC-0159-containing LNP-mRNA (mRNA dose typically 1-50 μg/mouse) via intramuscular (hind leg) or intravenous (tail vein) injection. 3) Collect blood and dissect major tissues (muscle, lymph nodes, liver, spleen) at multiple time points post-administration (e.g., 6h, 24h, 48h, 72h). 4) Observe tissue distribution of fluorescently labeled mRNA using in vivo imaging systems. 5) Measure specific antibody titers in serum by ELISA, and detect T cell responses by ELISpot or flow cytometry. 6) For pharmacokinetic studies, quantify ALC-0159 concentrations in plasma and tissues by LC-MS/MS. Studies show that after intramuscular injection of 5 mg/kg ALC-0159, the peak concentration Cmax is approximately 3.4 μg/mL, and the elimination half-life t₁/₂ is approximately 130.9 hours.

The pharmacokinetics of ALC-0159 have been systematically studied in rats. This PEG-lipid exhibits extremely slow elimination from plasma. Following intramuscular injection of 5 mg/kg, key pharmacokinetic parameters are: peak concentration Cmax 3.415±0.794 μg/mL; elimination half-life t₁/₂ 130.9±60.671 hours; clearance CL 0.024±0.006 L/h/kg; apparent volume of distribution Vd 4.27±1.476 L/kg; area under the curve AUC(0-t) 181.061±36.533 mg/L·h; mean residence time MRT(0-∞) 133.645±43.629 hours. The 10 mg/kg dose group shows similar slow elimination characteristics, with t₁/₂ approximately 103.7 hours. Excretion studies show that ALC-0159 is primarily excreted unchanged in feces, with a cumulative fecal excretion rate of approximately 72.0% of the administered dose within 120 hours, while urinary levels are below the lower limit of quantification.

The toxicological concerns for ALC-0159 as a PEG-lipid primarily center on its potential immunogenicity and hypersensitivity reactions. PEG-based compounds are known to induce anti-PEG antibodies in some individuals, potentially leading to allergic reactions. Between December 2020 and August 2021, approximately 5,805 anaphylaxis reports (approximately 10.44 per million doses) were documented following administration of about 560 million doses of Comirnaty® vaccine in the United States and Europe, with ALC-0159 speculated to be a potential sensitizing excipient. Additionally, when ALC-0159 is covalently conjugated to siRNA and accumulates at high levels in tissues (e.g., >20 pmol/mg tissue), non-specific modulation of gene expression may be observed, indicating potential risks at high doses or with specific delivery strategies. In in vitro cell assays, LNPs containing ALC-0159 show no significant cytotoxicity at therapeutic concentrations.

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

ALC-0315 is an ionizable amino lipid responsible for mRNA compression and promotes mRNA delivery and cytoplasmic release through a hypothetical endosome destabilization mechanism. The lipid nanoparticles (LNPs) in the Pfizer-BioNTech COVID-19 vaccine contain a low concentration (<2 mol%) of ALC-0159, which contributes to the stability of the nanoparticles through the steric hindrance mechanism of its polyethylene glycol (PEG) moiety. Given the low ALC-0159 content, the LNPs in the Pfizer-BioNTech COVID-19 vaccine likely only exhibit weak PEG steric hindrance. [1]

---

See Figure 1, where the red arrow indicates the safe termination point.
Basic Protocol 1: In Vitro Transcription and Enzymatic Capping and Tailing of mRNA
Completing the entire protocol (including the preparation and evaluation of capped and tailed mRNAs) takes 2 to 4 days. If overnight precipitation is planned, it takes 4 days. The in vitro transcription, capping, and tailing reactions take about half a day. All reaction preparation can be completed in approximately 1 hour, followed by the required incubation times: 2 hours for in vitro transcription and 1 hour for capping/tailing. mRNA precipitation requires a 30-minute centrifugation step, and RNA resuspending requires approximately 10 minutes. Quality assessment of mRNA using Nanodrop, agarose gel, and automated gel electrophoresis requires 1 hour.
Note: When scaling up experiments, more time is required, especially when multiple or large mRNA precipitates need to be resuspended in nuclease-free water.
Basic Protocol 2: Encapsulating mRNA in iLNP
Lipid solution preparation can be performed before the formulation step. Otherwise, the entire encapsulation protocol must be completed on the same day. All reagents should be allowed to reach room temperature for 1 hour before use, and preparation and dilution in DPBS also require 1 hour. The centrifugation concentration step time depends on particle size, total sample volume, and desired final volume. It typically takes 1 to 4 hours. Concentrated iLNP solutions can be stored in a refrigerator until the required dilution is determined by RiboGreen assay. Alternative: Small-scale mRNA encapsulation using pre-prepared vesicles. Same as Basic Protocol 2. Basic Protocol 3: Characterization and Quality Control of mRNA iLNPs. RiboGreen assays require 1 to 2 hours, including time for the kit to warm to room temperature after removal from the refrigerator. DLS (particle size, PDI, zeta potential) analysis should be performed on the final diluted samples before biological evaluation. Sample preparation takes several minutes, and analysis time varies depending on the instrument, but typically requires 5 to 10 minutes per sample. The buffer used in TNS assays must reach room temperature before use. This may take several hours depending on the aliquot size. The buffer can be refrigerated the night before assay to reduce preheating time. Each iLNP sample requires 40 aliquots in a 96-well plate, therefore each iLNP sample requires 10 to 15 minutes of plate preparation time and 10 minutes of plate reading time.
The mRNA extraction step takes 30 minutes. For automated gel electrophoresis analysis of the extracted mRNA, please refer to the above (Basic Protocol 1). [3]

(C2H4O)NC31H63NO2

1849616-42-7

155977658

White to off-white solid

1

4

31

38

415

0

O=C(C([H])([H])OC([H])([H])C([H])([H])OC([H])([H])[H])N(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H].N([H])([H])[H]

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (Infinity mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)