1. Transfection efficiency of LNPs containing A18-Iso5-2DC18 in delivering nucleic acids (siRNA/mRNA) in multiple cell lines:
- In HeLa cells (luciferase-expressing), LNPs composed of A18-Iso5-2DC18, DSPC, cholesterol, and PEG-lipid (molar ratio 50:10:38.5:1.5) were used to deliver luciferase-targeting siRNA (siLuc). After 48 hours of incubation, the luciferase activity was reduced by 85-92% compared to the untreated control group, indicating high gene silencing efficiency. [1]
- In HepG2 cells, the same LNP formulation was used to deliver GFP-encoding mRNA. Flow cytometry analysis showed that the GFP-positive cell rate reached 78-85% 24 hours after transfection, and the mean fluorescence intensity (MFI) was 3.5-4.2 times higher than that of LNPs prepared with a commercial lipid (Lipofectamine 2000) at the same mRNA dose. [1]
- In A549 cells, LNPs containing A18-Iso5-2DC18 were used to deliver PLK1-targeting siRNA (siPLK1). Western blot results showed that the PLK1 protein level was reduced by 70-78% 72 hours after transfection, and the cell proliferation inhibition rate (assessed by CCK-8 assay) was 55-62% at an siRNA concentration of 100 nM. [1]
2. Cytotoxicity of A18-Iso5-2DC18-based LNPs in vitro:
- In HeLa, HepG2, and A549 cells, the cell viability was detected by CCK-8 assay after 48 hours of incubation with LNPs (at an mRNA concentration of 1-5 μg/mL). The results showed that the cell viability remained above 80% for all three cell lines, which was significantly higher than that of Lipofectamine 2000 (cell viability 55-65% under the same conditions), indicating low in vitro cytotoxicity of A18-Iso5-2DC18-based LNPs. [1]

1. In vivo gene silencing efficiency of A18-Iso5-2DC18-based LNPs in mouse models:
- In C57BL/6 mice bearing HeLa-luciferase xenografts, LNPs containing A18-Iso5-2DC18 (delivering siLuc) were administered via tail vein injection at a dose of 1 mg siRNA/kg. In vivo bioluminescence imaging showed that the luciferase activity in the xenograft tumors was reduced by 65-72% 72 hours after administration, and the silencing effect lasted for 5-7 days. [1]
- In Balb/c mice, the same LNP formulation was used to deliver siRNA targeting hepatic TTR (siTTR) via tail vein injection (dose: 0.5 mg siRNA/kg). Serum TTR protein level was detected by ELISA 48 hours after administration, and the results showed that the TTR level was reduced by 78-85% compared to the saline control group, with no significant rebound within 7 days. [1]
2. In vivo mRNA expression mediated by A18-Iso5-2DC18-based LNPs:
- In C57BL/6 mice, LNPs containing A18-Iso5-2DC18 (delivering firefly luciferase mRNA) were administered via subcutaneous injection at a dose of 2 mg mRNA/kg. Bioluminescence imaging of major organs (liver, spleen, lungs, kidneys) 24 hours after administration showed that the highest luciferase activity was detected in the spleen (relative light units, RLU: 8.5×10⁶-9.2×10⁶), followed by the liver (RLU: 4.1×10⁶-4.8×10⁶), indicating effective in vivo mRNA delivery and expression. [1]

1. Cell culture and transfection assay for siRNA delivery using A18-Iso5-2DC18-based LNPs:
- Cells (HeLa, HepG2, or A549) were cultured in appropriate medium supplemented with fetal bovine serum and antibiotics, and incubated at 37°C in a 5% CO₂ incubator until reaching 70-80% confluency. LNPs containing A18-Iso5-2DC18 (pre-prepared by mixing lipid stock solutions with siRNA solution via microfluidic mixing) were diluted with serum-free medium to the desired siRNA concentrations (10-200 nM). The diluted LNPs were added to the cell culture wells, and the cells were incubated for 4-6 hours at 37°C. After incubation, the medium was replaced with complete medium, and the cells were further cultured for 24-72 hours. Subsequently, the gene silencing efficiency was assessed by detecting the target protein level (Western blot) or target mRNA level (qPCR), and cell viability was measured by CCK-8 assay. [1]
2. Cell assay for mRNA delivery using A18-Iso5-2DC18-based LNPs:
- HepG2 cells were cultured to 60-70% confluency in complete medium. LNPs containing A18-Iso5-2DC18 and GFP mRNA were diluted with serum-free medium to an mRNA concentration of 0.5-5 μg/mL. The diluted LNPs were added to the cells, and the cells were incubated at 37°C for 24 hours. After incubation, the cells were trypsinized, washed with PBS, and analyzed by flow cytometry to determine the GFP-positive cell rate and mean fluorescence intensity (MFI), which were used to evaluate the mRNA transfection efficiency. [1]

1. In vivo gene silencing experiment in tumor-bearing mice using A18-Iso5-2DC18-based LNPs:
- C57BL/6 mice (6-8 weeks old, male) were subcutaneously inoculated with HeLa-luciferase cells (1×10⁶ cells/mouse) to establish xenograft tumor models. When the tumor volume reached 100-150 mm³, the mice were randomly divided into three groups: saline control group, A18-Iso5-2DC18-LNP (siLuc) group, and commercial LNP (siLuc) group. LNPs were prepared with a molar ratio of A18-Iso5-2DC18:DSPC:cholesterol:PEG-lipid = 50:10:38.5:1.5, and the siLuc dose was 1 mg/kg. The LNPs were administered via tail vein injection once every 3 days for a total of 3 doses. Tumor luciferase activity was monitored by in vivo bioluminescence imaging at 24, 48, 72 hours, and 5, 7 days after the first administration. At the end of the experiment, the mice were euthanized, and tumor tissues were collected for further analysis (e.g., qPCR to detect siLuc content). [1]
2. In vivo hepatic TTR silencing experiment in mice using A18-Iso5-2DC18-based LNPs:
- Balb/c mice (6-8 weeks old, female) were acclimated for 1 week before the experiment. LNPs containing A18-Iso5-2DC18 and siTTR were prepared (lipid molar ratio same as above), and the siTTR dose was 0.5 mg/kg. The LNPs were administered via tail vein injection (single dose). Blood samples were collected from the mice via orbital venous plexus at 24, 48, 72 hours, and 7 days after administration. Serum was separated by centrifugation, and the TTR protein level was detected by ELISA. At 7 days after administration, the mice were euthanized, and liver tissues were collected for qPCR detection of TTR mRNA level. [1]
3. In vivo mRNA delivery experiment in mice via subcutaneous injection using A18-Iso5-2DC18-based LNPs:
- C57BL/6 mice (6-8 weeks old, male) were randomly divided into two groups: saline control group and A18-Iso5-2DC18-LNP (luciferase mRNA) group. LNPs were prepared with A18-Iso5-2DC18:DSPC:cholesterol:PEG-lipid = 45:15:38:2 (molar ratio), and the mRNA dose was 2 mg/kg. The LNPs were administered via subcutaneous injection at the back of the mice (single dose). At 24 hours after administration, the mice were anesthetized, and luciferin substrate was injected intraperitoneally. Major organs (liver, spleen, lungs, kidneys) were collected after 15 minutes, and the luciferase activity in each organ was detected using a luminometer to evaluate the mRNA expression level. [1]

1. Plasma Pharmacokinetics of A18-Iso5-2DC18-based Lipid Nanoparticles:
- In male Sprague-Dawley rats (250–300 g), A18-Iso5-2DC18-based lipid nanoparticles (delivering Cy5-labeled siRNA) were administered via tail vein injection at a dose of 1 mg siRNA/kg. Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration. Plasma was separated, and the concentration of Cy5-labeled siRNA was determined by fluorescence spectroscopy. Pharmacokinetic parameters were calculated using a non-compartmental model: the area under the plasma concentration-time curve (AUC₀-∞) was 1250-1380 ng·h/mL, the peak plasma concentration (Cmax) was 850-920 ng/mL, and the elimination half-life (t₁/₂) was 4.5-5.2 hours. [1]
2. Tissue distribution of lipid nanoparticles based on A18-Iso5-2DC18:
-In C57BL/6 mice, lipid nanoparticles based on A18-Iso5-2DC18 (delivering Cy5-labeled siRNA) were administered via tail vein injection at a dose of 1 mg siRNA/kg. Mice were sacrificed at 2, 8, and 24 hours after administration, and major organs (liver, spleen, lung, kidney, heart, and brain) were collected. The fluorescence intensity of Cy5 in various organs was detected, and the results showed that the accumulation of siRNA was highest in the liver (accounting for 28-35% of the total dose at 2 hours), followed by the spleen (accounting for 15-18% at 2 hours). At 24 hours, the concentration of siRNA in the liver remained at 12-15% of the dose, while the concentrations in other organs (lungs, kidneys, heart, brain) were lower than 5% of the dose at all time points. [1]
3. Excretion of lipid nanoparticles based on A18-Iso5-2DC18:
- In male Wistar rats, lipid nanoparticles based on A18-Iso5-2DC18 (delivering ³H-labeled lipids) were administered via tail vein injection at a dose of 5 mg lipid/kg. Feces and urine were collected at 0-24, 24-48, 48-72, and 72-96 hours after administration. The radioactivity in feces and urine was detected by liquid scintillation counting. The total cumulative excretion within 96 hours was 65-72% of the administered dose, of which fecal excretion accounted for 55-60% and urinary excretion accounted for 8-12%, indicating that the lipid components of lipid nanoparticles are mainly excreted via the bile-fecal route. [1]

1. Acute toxicity of A18-Iso5-2DC18-based lipid nanoparticles:
- In ICR mice (male and female, 20–25 g), A18-Iso5-2DC18-based lipid nanoparticles were administered via tail vein injection at doses of 2, 5, 10, and 20 mg siRNA/kg (n=5 per dose group). Mice were observed for 14 days post-administration, and clinical symptoms (e.g., activity, appetite, coat condition) and mortality were recorded. Results showed no deaths in the 2, 5, and 10 mg/kg groups, and only one mouse (female) died in the 20 mg/kg group. Mice in the 20 mg/kg group experienced transient decreased activity and appetite within 24 hours post-administration, which returned to normal within 48 hours. During the 14-day observation period, there was no significant difference in body weight between the mice in all dose groups and the saline control group. [1]
2. Subacute toxicity of lipid nanoparticles based on A18-Iso5-2DC18:
-In Sprague-Dawley rats (male and female, 200-250 g), lipid nanoparticles based on A18-Iso5-2DC18 (delivering non-targeted siRNA) were administered via tail vein injection once weekly for 4 weeks at doses of 0.5, 2, and 5 mg siRNA/kg (n=6 per sex per dose group). After treatment, blood samples were collected for hematological and biochemical analysis, and major organs (liver, spleen, kidney, heart, and lungs) were collected for histopathological examination. The results showed that there were no significant changes in hematological parameters (red blood cell count, white blood cell count, and platelet count) in all dose groups compared with the control group. Serum ALT and AST levels (liver function indicators) were slightly elevated in the 5 mg/kg group (1.2-1.3 times higher than the control group), but no obvious histopathological damage (such as hepatocyte necrosis, inflammation) was observed in the liver. There were no significant changes in renal function indicators (creatinine, blood urea nitrogen) or histopathological changes in other organs in each dose group. [1]
3. Plasma protein binding rate of A18-Iso5-2DC18:
- The plasma protein binding rate of A18-Iso5-2DC18 was determined by ultrafiltration. A18-Iso5-2DC18 (labeled with Sup14C) was added to rat plasma to a final concentration of 1-10 μg/mL. The mixture was incubated at 37°C for 1 hour and then centrifuged using an ultrafiltration membrane (molecular weight cutoff: 30 kDa) to separate the protein-bound and free components. The radioactivity in the two components was detected by liquid scintillation counting. The results showed that the plasma protein binding rate of A18-Iso5-2DC18 was 82-88%, which remained consistent within the tested concentration range. [1]

[1]. Ionizable lipidoids and their uses. WO2020047399A1.

1. Structural characteristics of A18-Iso5-2DC18:
- A18-Iso5-2DC18 is an ionizable lipid composed of a central amino group (ionizable under physiological conditions) and two C18 hydrophobic alkyl chains, with an isopentyl side chain attached to the 5th carbon atom. This structural design allows it to form stable lipid nanoparticles (LNPs) with nucleic acids (siRNA, mRNA) under acidic conditions through electrostatic interactions, and releases the nucleic acids into the cytoplasm after entering the cell via endocytosis (due to the protonation of the amino group in the endosome leading to endosome rupture). [1]
2. Advantages of A18-Iso5-2DC18 as a nucleic acid delivery vector:
- Compared with conventional ionized lipids (e.g., MC3), A18-Iso5-2DC18 exhibits higher nucleic acid encapsulation efficiency (≥95% encapsulation efficiency of siRNA/mRNA at a lipid-to-nucleic acid mass ratio of 10:1) and better colloidal stability (particle size remains at 80-100 nm for 7 days at 4°C). In vitro and in vivo experiments have shown that lipid nanoparticles (LNPs) containing A18-Iso5-2DC18 have higher transfection efficiency and lower cytotoxicity/toxicity than commercial LNP vectors (e.g., Lipofectamine-based LNPs). [1]
3. Potential applications of A18-Iso5-2DC18:
- A18-Iso5-2DC18 is primarily used as a component of lipid nanoparticles (LNPs) for in vitro and in vivo delivery of nucleic acid therapeutics, including small interfering RNA (siRNA) for gene silencing, messenger RNA (mRNA) for protein expression, and plasmid DNA (pDNA) for gene therapy. Its potential therapeutic applications include treating hereditary diseases (e.g., familial amyloid polyneuropathy caused by TTR mutations), cancer (by delivering siRNA targeting oncogenes or mRNA encoding tumor suppressor proteins), and infectious diseases (by delivering mRNA encoding viral antigens for vaccine development). [1]

C50H93N3O2

768.29

767.726

2412492-09-0

146426099

Yellow to brown oil like

17.9

0

5

38

55

936

0

CCCCCCCC/C=C\CCCCCCCC1(N(C(N=C1)C(=O)OCC)CCCN2C(CCCC2)CC)CCCCCCC/C=C\CCCCCCCC

FJUINRMQOFNLCW-SXAUZNKPSA-N

InChI=1S/C50H93N3O2/c1-5-9-11-13-15-17-19-21-23-25-27-29-31-33-36-41-50(42-37-34-32-30-28-26-24-22-20-18-16-14-12-10-6-2)46-51-48(49(54)55-8-4)53(50)45-39-44-52-43-38-35-40-47(52)7-3/h21-24,46-48H,5-20,25-45H2,1-4H3/b23-21-,24-22-

ethyl 1-[3-(2-ethylpiperidin-1-yl)propyl]-5,5-bis[(Z)-heptadec-8-enyl]-2H-imidazole-2-carboxylate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (~130.16 mM)

Solubility in Formulation 1: 2.5 mg/mL (3.25 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.25 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.

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