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| Targets |
None (excipient). C14-4 is an ionizable lipid that complexes with mRNA and forms LNPs, facilitating delivery of CAR mRNA into primary human T cells. It enables efficient transfection of hard-to-transfect cells such as T cells, leading to CAR expression on the T cell surface.
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| ln Vitro |
C14-4 LNPs efficiently encapsulate CAR mRNA (>90%). In vitro, C14-4 LNPs transfect primary human T cells with high efficiency (typically 30-60% CAR-positive T cells). C14-4 LNPs enable transient CAR expression peaking at 24-48 h post-transfection. CAR-T cells generated with C14-4 LNPs exhibit potent cytotoxicity against target cancer cells (e.g., CD19-positive Raji cells) in co-culture assays, as measured by target cell lysis (LDH release) and cytokine production (IFN-gamma, IL-2).
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| ln Vivo |
C14-4 LNPs have been evaluated for in vivo generation of CAR-T cells. Intravenous administration of C14-4 LNPs encapsulating anti-CD19 CAR mRNA to immunocompetent mice leads to CAR expression on T cells in vivo. These in vivo-generated CAR-T cells effectively clear CD19-positive tumor cells, resulting in reduced tumor burden and prolonged survival. C14-4 LNPs show low systemic toxicity and favorable biodistribution.
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| Enzyme Assay |
C14-4 (C₈4H1₇3N₅O₇, MW 1365.30) is a light yellow to yellow liquid. For LNP preparation, C14-4 is dissolved in ethanol and mixed with helper lipids (e.g., DSPC, cholesterol, PEG2000-DMG) at optimized molar ratios (e.g., 50:10:38.5:1.5). The mixture is combined with an acidic aqueous buffer (25-50 mM sodium acetate, pH 4-5) containing CAR mRNA via microfluidic mixing. LNPs (size 80-120 nm) form spontaneously. Ethanol is removed by dialysis or TFF, and pH is adjusted to 7.4. Encapsulation efficiency (>90%) is measured by Ribogreen assay. Particle size, PDI (0.1-0.2), and zeta potential (∼+10 to +30 mV at pH 5, ∼0 mV at pH 7.4) are characterized by DLS. pKa (∼6.5-6.8) is determined by TNS fluorescence.
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| Cell Assay |
For in vitro studies, primary human T cells are isolated from peripheral blood mononuclear cells (PBMCs) using CD3+ magnetic bead separation. T cells are activated with anti-CD3/CD28 beads or antibodies in the presence of IL-2 (100-200 IU/mL) for 24-48 h. Activated T cells are seeded in 96- or 24-well plates (1-2×10⁵ cells/well) in serum-free medium (e.g., X-VIVO15) and treated with C14-4 LNPs at CAR mRNA doses of 0.5-5 ug/1×10⁶ cells. After 4-6 h, the LNP-containing medium is replaced with fresh medium containing IL-2. CAR expression is assessed by flow cytometry using protein L or anti-F(ab')2 antibody staining at 24-48 h post-transfection. Cytotoxicity is evaluated by co-culturing CAR-T cells (effector) with target cells (e.g., Raji, K562) at E:T ratios (1:1 to 20:1) for 4-24 h, followed by LDH release assay or flow cytometry-based killing assay. Cytokine production (IFN-gamma, IL-2, TNF-alpha) is measured by ELISA.
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| Animal Protocol |
For in vivo studies, C14-4 LNPs encapsulating anti-CD19 CAR mRNA are administered intravenously via the tail vein to immunocompetent mice (e.g., BALB/c or C57BL/6, 6-8 weeks old) at mRNA doses of 0.2-2 mg/kg. For tumor models: mice are injected i.v. with CD19-positive tumor cells (e.g., A20, Raji, 1-5×10⁵/mouse) 3-7 days before LNP treatment. LNPs are administered on days 1, 3, 5 (multiple doses). Tumor burden is assessed by bioluminescence imaging (if tumor cells express luciferase) or by measuring tumor volume (for subcutaneous models). Survival is monitored. For CAR-T generation quantification: peripheral blood is collected at various time points, and CAR expression on T cells is measured by flow cytometry. Tissues (spleen, liver, bone marrow) are harvested for CAR expression analysis. Biodistribution: fluorescently labeled LNPs are used, and tissues are imaged ex vivo. Cytokine release syndrome (CRS) markers (IL-6, IFN-gamma, TNF-alpha) in plasma are measured by ELISA.
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| ADME/Pharmacokinetics |
No detailed PK data for C14-4 has been reported. C14-4 LNPs have a circulation half-life of ∼2-4 hours following i.v. administration, with predominant accumulation in the liver (∼40-60% of dose) and spleen (∼20-30%). The ionizable lipid has a pKa of ∼6.5-6.8, enabling efficient endosomal escape. Metabolism likely involves hydrolysis of ester linkages, followed by clearance of lipid fragments via biliary and renal routes. The high molecular weight (1365.30 Da) suggests low oral bioavailability.
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| Toxicity/Toxicokinetics |
C14-4 LNPs have high transfection efficiency and low cytotoxicity (cell viability >85% in primary human T cells at 2.5 ug/mL mRNA). In vivo, C14-4 LNPs are well-tolerated at CAR mRNA doses ≤1 mg/kg i.v. Mild, transient elevation of liver enzymes (ALT/AST) and pro-inflammatory cytokines (IL-6, IFN-gamma) may occur, which is typical of LNP administration. No severe adverse events (e.g., neurotoxicity, CRS) have been reported at therapeutic doses.
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| References | |
| Additional Infomation |
C14-4 (C14-494, CAS 2639634-80-1, C₈4H1₇3N₅O₇, MW 1365.30) has ≥98% purity and is a light yellow to yellow liquid. Storage at -20degC is required. C14-4 is an ionizable lipid used for CAR mRNA delivery to T cells. No clinical trials have been reported; it is for research use only. C14-4 enables in vivo generation of CAR-T cells, offering a simpler and potentially safer alternative to ex vivo CAR-T manufacturing. This technology is in preclinical development.
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| Molecular Formula |
C84H173N5O7
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|---|---|
| Molecular Weight |
1365.30
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| Exact Mass |
1364.333
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| CAS # |
2639634-80-1
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| PubChem CID |
164687424
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| Appearance |
Light yellow to yellow liquid
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| LogP |
27.4
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
80
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| Heavy Atom Count |
96
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| Complexity |
1420
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CCCCCCCCCCCCC(CN(CCN1CCN(CC1)CCOCCN(CC(CCCCCCCCCCCC)O)CC(CCCCCCCCCCCC)O)CCOCCN(CC(CCCCCCCCCCCC)O)CC(CCCCCCCCCCCC)O)O
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| InChi Key |
OQALBVGFAKJDAR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C84H173N5O7/c1-6-11-16-21-26-31-36-41-46-51-56-80(90)75-87(68-72-96-74-70-89(78-83(93)59-54-49-44-39-34-29-24-19-14-9-4)79-84(94)60-55-50-45-40-35-30-25-20-15-10-5)66-65-85-61-63-86(64-62-85)67-71-95-73-69-88(76-81(91)57-52-47-42-37-32-27-22-17-12-7-2)77-82(92)58-53-48-43-38-33-28-23-18-13-8-3/h80-84,90-94H,6-79H2,1-5H3
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| Chemical Name |
1-[2-[2-[bis(2-hydroxytetradecyl)amino]ethoxy]ethyl-[2-[4-[2-[2-[bis(2-hydroxytetradecyl)amino]ethoxy]ethyl]piperazin-1-yl]ethyl]amino]tetradecan-2-ol
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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) |
DMSO: 100 mg/mL (73.24 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (1.83 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.7324 mL | 3.6622 mL | 7.3244 mL | |
| 5 mM | 0.1465 mL | 0.7324 mL | 1.4649 mL | |
| 10 mM | 0.0732 mL | 0.3662 mL | 0.7324 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
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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