| Targets |
244cis targets the endosomal membrane after LNP‑mediated cellular uptake. As an ionizable lipid, it has a low pKa (approx. 6.5). At physiological pH (7.4), it is neutrally charged, minimizing non‑specific interactions and toxicity. Upon endocytosis, the acidic environment of the endosome (pH 5.5-6.0) protonates the piperazine nitrogen, giving the lipid a positive charge. This positive charge destabilizes the endosomal membrane, allowing the encapsulated mRNA to escape into the cytoplasm. The lipid also targets the lung tissue after intravenous administration.
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| ln Vitro |
In vitro, LNPs formulated with 244cis are used to transfect cells with high efficiency. For example, LNPs containing 244cis, cholesterol, DSPC, and PEG‑lipid are prepared by a rapid mixing method. The LNPs are loaded with mRNA encoding luciferase or GFP. HEK293 cells are seeded in 96‑well plates and treated with the LNPs (0.1-10 microg mRNA/mL). After 24 h, transfection efficiency is assessed by measuring luciferase activity or GFP fluorescence. The 244cis LNPs show higher transfection efficiency and lower cytotoxicity compared to other ionizable lipids.
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| ln Vivo |
In vivo, 244cis LNPs accumulate specifically in the lungs of mice after intravenous injection. A mouse study compared LNPs containing 244cis with LNPs containing SM‑102, the ionizable lipid used in the Moderna COVID‑19 vaccine. LNPs were loaded with an mRNA reporter gene (e.g., luciferase). BALB/c mice were injected intravenously with 0.5 mg/kg mRNA. Bioluminescence imaging at 6 h showed that 244cis LNPs induced high luciferase expression specifically in the lungs, whereas SM‑102 LNPs induced expression mainly in the liver. Compared with LNPs containing SM-102, LNPs containing 244cis and coated with an mRNA reporter gene accumulated specifically in the lungs of mice and induced a decrease in serum chemokine (CC motif) ligand 2 (CCL2) levels.
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| Enzyme Assay |
The pKa of 244cis is determined by a TNS (2‑(p‑toluidinyl)naphthalene‑6‑sulfonic acid) fluorescence assay. The lipid is dispersed in buffers of varying pH (3-10). TNS is added, and the fluorescence is measured (ex 322 nm, em 432 nm). The pKa is calculated from the inflection point of the fluorescence vs. pH curve. 244cis typically has a pKa of around 6.5. The encapsulation efficiency of mRNA in LNPs is measured using the Quant‑iT RiboGreen RNA assay. LNPs are incubated with RiboGreen dye in the presence or absence of 1% Triton X‑100. The difference in fluorescence (ex 485 nm, em 535 nm) gives the amount of encapsulated RNA.
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| Cell Assay |
HEK293 cells are seeded in 96‑well plates at 2×10⁴ cells/well. LNPs (244cis) loaded with luciferase mRNA are added to the cells at serial dilutions (0.01-10 microg mRNA/mL). After 24 h, the cells are lysed, and luciferase activity is measured by adding the luciferin substrate and reading the luminescence in a plate reader. A standard curve using a luciferase protein standard is used to calculate the amount of luciferase produced per cell. Cell viability is assessed using the CellTiter‑Glo assay to ensure that the LNPs are not toxic to the cells.
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| Animal Protocol |
For the in vivo study, female BALB/c mice (6-8 weeks old, n=5 per group) are injected intravenously via the tail vein with 200 microL of LNPs containing 0.5 mg/kg mRNA encoding firefly luciferase. At 6 h post‑injection, the mice are injected intraperitoneally with D‑luciferin (150 mg/kg). After 10 min, the mice are anesthetized and imaged using an IVIS Spectrum to measure bioluminescence. The mice are then euthanized. The lungs, liver, spleen, heart, and kidneys are harvested and ex vivo imaging is performed. The total flux (photons/sec) in the lung region is measured. The serum CCL2 levels are measured by ELISA.
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| ADME/Pharmacokinetics |
244cis is an ionizable cationic lipid. Pharmacokinetic properties of the lipid itself are not directly measured; instead, the biodistribution of the LNP is measured by labeling the lipid with a fluorescent dye (e.g., DiD). After injection into mice, LNPs are cleared from the circulation with a half‑life of approximately 2-4 h. Biodistribution analysis shows that 244cis LNPs accumulate primarily in the lungs (50‑60% of injected dose per gram) and secondarily in the liver and spleen. The lipid is metabolized in the liver and excreted via the bile. The mRNA cargo is degraded by nucleases.
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| Toxicity/Toxicokinetics |
The toxicity of 244cis has been evaluated in mice. In the acute toxicity study described above, no significant weight loss or behavioral changes are observed after a single intravenous injection of 0.5 mg/kg mRNA. Histological analysis of the lungs at 48 h post‑injection shows minimal inflammation. The low immunogenicity of 244cis allows for repeated dosing without loss of activity. The compound is for research use only; its chronic toxicity and carcinogenicity have not been evaluated.
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| References | |
| Additional Infomation |
244cis is not an approved drug. It is a research‑only ionizable lipid for the formulation of LNPs for mRNA delivery. It is specifically developed for the treatment of chronic diseases in the lung with improved potency and safety. This lipid enables the repeated dosing of mRNA without the loss of activity due to its low immunogenicity. 244cis is used in the development of inhaled mRNA therapeutics for conditions such as cystic fibrosis, pulmonary fibrosis, and lung cancer. It is not for human use.
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| CAS # |
2956402-64-3
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| Appearance |
Colorless to light yellow liquid
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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