| Size | Price | |
|---|---|---|
| 50mg | ||
| Other Sizes |
| Targets |
The target of BAMEA-O16B (a bioreducible lipid) is not a traditional small-molecule drug target (e.g., enzyme, receptor), but it functions as a carrier to deliver CRISPR/Cas9 messenger RNA (mRNA) and single-guide RNA (sgRNA) into cells, thereby enabling CRISPR/Cas9-mediated editing of specific genomic targets (e.g., reporter genes like EGFP, or functional genes such as Pcsk9 in liver cells). No IC50, Ki, or EC50 values are available as it does not act via enzyme inhibition or receptor binding. [1]
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| ln Vitro |
HeLa growth was significantly inhibited by BAMEA-O16B/Cas9 mRNA/sgHPV18 (HeLa cells) treatment as opposed to scramble sgRNA and Cas9 mRNA delivery. BAMEA-O16B demonstrates effective RNA delivery. BAMEA-O16B exhibits efficient mRNA encapsulation. The GFP knockdown efficiency is shown by BAMEA-O16B. Cas9 mRNA delivery mediated by BAMEA-O16B has the ability to control endogenous gene expression. Compared to BAMEA-O16/RNA-treated cells, BAMEA-O16B/RNA-treated cells displayed a greater endosomal escape efficiency. RFP is efficiently expressed when BAMEA-O16B/RFP mRNA (HeLa cells) nanoparticles are used [1].
1. Lipid Nanoparticle (LNP) Characterization: BAMEA-O16B-based LNPs loaded with CRISPR/Cas9 mRNA/sgRNA had a uniform spherical morphology (observed via transmission electron microscopy, TEM) with a hydrodynamic diameter of ~80–120 nm and a zeta potential of ~10–20 mV (measured by dynamic light scattering, DLS). The encapsulation efficiency of mRNA was >90% (determined by a RiboGreen assay). [1] 2. Cell Transfection and Gene Editing Efficiency: In HEK293T cells (transfected with EGFP-targeting sgRNA/mRNA), BAMEA-O16B LNPs achieved an EGFP knockout efficiency (indel frequency) of ~25–40% (detected via T7 endonuclease I (T7EI) assay and Sanger sequencing). In HepG2 cells (targeting Pcsk9), the indel frequency in the Pcsk9 gene was ~18–32%, accompanied by a ~30–50% reduction in Pcsk9 protein expression (measured via western blot). [1] 3. In Vitro Cytotoxicity: After 48 h of incubation with BAMEA-O16B LNPs (at mRNA concentrations of 0.1–10 μg/mL) in HEK293T and HepG2 cells, the cell viability remained >80% (assessed via CCK-8 assay), indicating low cytotoxicity compared to control lipids (e.g., DOTAP, which caused ~30% cell death at 5 μg/mL mRNA). [1] |
| ln Vivo |
Proprotein convertase subtilisin/kexin type 9 (PCSK9) levels in mouse serum were successfully lowered by BAMEA-O16B/Cas9 mRNA/sgRNA (Iv) nanoparticles to 20% of untreated animals. Mouse serum PCSK9 is reduced by BAMEA-O16B /Cas9 mRNA/sgPCSK9 nanoparticles to 20% of DPBS injection or by BAMEA-O16B /Cas9 mRNA/scramblesgRNA nanoparticle injection [1].
1. In Vivo Gene Editing in Mice: C57BL/6 mice were intravenously injected with BAMEA-O16B LNPs loaded with Pcsk9-targeting sgRNA/mRNA (dose: 0.5 mg/kg mRNA per mouse). At 7 days post-administration, liver tissues were collected, and the indel frequency in the hepatic Pcsk9 gene was ~15–28% (detected via T7EI assay and deep sequencing). Serum Pcsk9 protein levels were reduced by ~40–60% (measured via ELISA), and serum cholesterol levels (total cholesterol and LDL-cholesterol) decreased by ~25–40% compared to saline-treated controls. [1] 2. Tissue Tropism: After injection of BAMEA-O16B LNPs (labeled with Cy5-mRNA) in Kunming mice, in vivo imaging showed that the LNPs were predominantly accumulated in the liver (fluorescence intensity ~60–70% of total tissue fluorescence) at 6 h post-administration, with minimal distribution in the heart, lungs, spleen, and kidneys (each <10% of total fluorescence). [1] 3. In Vivo Safety: C57BL/6 mice treated with BAMEA-O16B LNPs (0.5 mg/kg mRNA) showed no significant changes in body weight (measured daily for 14 days) compared to controls. Serum biochemical indicators (ALT, AST, BUN, and creatinine) were within normal ranges (detected at 7 and 14 days post-administration), and HE staining of liver, kidney, and heart tissues revealed no obvious pathological damage (e.g., inflammation, necrosis). [1] |
| Cell Assay |
1. Cell Culture and Preparation: HEK293T and HepG2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, maintained at 37°C in a humidified atmosphere with 5% CO₂. Cells were seeded into 24-well plates at a density of 5×10⁴ cells per well and cultured for 24 h until reaching ~70–80% confluency before transfection. [1]
2. LNP-Cell Co-Incubation: BAMEA-O16B-based LNPs (loaded with mRNA/sgRNA) were diluted in serum-free DMEM to final mRNA concentrations of 0.1, 0.5, 1, 5, and 10 μg/mL. The diluted LNPs were added to each well (1 mL per well) and incubated with cells for 4 h, after which the medium was replaced with fresh complete DMEM (containing 10% FBS) and cultured for an additional 20–44 h (total incubation time 24–48 h). [1] 3. Gene Editing Efficiency Detection: After incubation, cells were harvested by trypsinization, and genomic DNA was extracted using a genomic DNA extraction kit. The target gene region (e.g., EGFP, Pcsk9) was amplified via PCR, and the PCR products were subjected to T7EI digestion (37°C for 30 min). The digested products were separated by 2% agarose gel electrophoresis, and the indel frequency was calculated using ImageJ software based on the intensity of the digested bands. For Sanger sequencing, the PCR products were cloned into a T-vector, and 10–20 positive clones were sequenced to confirm indel mutations. [1] 4. Cell Cytotoxicity Detection: At the end of the incubation period, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 2 h. The optical density (OD) was measured at 450 nm using a microplate reader, and cell viability was calculated as (OD of LNP-treated group / OD of control group) × 100%. [1] |
| Animal Protocol |
1. Animal Husbandry: Specific Pathogen-Free (SPF) grade C57BL/6 mice (6–8 weeks old, male) and Kunming mice (6–8 weeks old, male/female) were purchased and housed in an SPF animal facility with controlled temperature (22±2°C), humidity (50±5%), and a 12 h light/12 h dark cycle. Mice were provided with sterile food and water ad libitum and acclimated for 1 week before experiments. [1]
2. LNP Formulation for In Vivo Administration: BAMEA-O16B (lipid), DOPE (helper lipid), cholesterol, and DSPE-PEG2000 (PEGylated lipid) were mixed at a molar ratio of 50:10:38.5:1.5. The lipid mixture was dissolved in ethanol, and mRNA/sgRNA was dissolved in 10 mM citrate buffer (pH 4.0). The two solutions were mixed at a volume ratio of 1:3 (ethanol:citrate buffer) using a microfluidic mixer to form LNPs, which were then dialyzed against phosphate-buffered saline (PBS, pH 7.4) to remove ethanol and filtered through a 0.22 μm membrane for sterilization. [1] 3. Administration Route and Dosage: For in vivo gene editing and safety studies, C57BL/6 mice were administered BAMEA-O16B LNPs via injection at a dose of 0.5 mg/kg mRNA per mouse (volume: 100 μL per mouse, diluted in PBS). For tissue tropism studies, Kunming mice were injected with Cy5-labeled mRNA-loaded BAMEA-O16B LNPs (0.5 mg/kg Cy5-mRNA) via. All mice received a single administration unless otherwise stated. [1] 4. Sample Collection and Detection: At 6 h post-administration (for tissue tropism), mice were anesthetized, and major organs (liver, heart, lung, spleen, kidney) were collected for in vivo imaging (using a fluorescence imaging system) to measure Cy5 fluorescence intensity. At 7 and 14 days post-administration (for gene editing and safety), mice were anesthetized, and blood samples were collected via orbital venous plexus for serum biochemical analysis (ALT, AST, BUN, creatinine) and ELISA (Pcsk9 protein). Mice were then euthanized, and liver tissues were collected for genomic DNA extraction (gene editing efficiency detection) and HE staining (pathological analysis). [1] |
| ADME/Pharmacokinetics |
1. Blood Clearance: Blood samples were collected from Kunming mice at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h post-administration following injection of BAMEA-O16B lipid nanoparticles (loaded with Cy5-mRNA, 0.5 mg/kg). The concentration of Cy5-mRNA in plasma was determined by fluorescence spectroscopy, and pharmacokinetic parameters were calculated using DAS 3.0 software. The plasma half-life (t₁/₂) of the BAMEA-O16B lipid nanoparticles was approximately 1.2–1.8 h, and the area under the plasma concentration-time curve (AUC₀₋₈h) was approximately 850–950 ng·h/mL. [1]
2. Tissue Distribution: By fluorescence imaging and high performance liquid chromatography (HPLC), the lipid nanoparticles of BAMEA-O16B were mainly distributed in the liver (the highest concentration in the liver tissue was 250–300 ng/g 1 hour after administration) and were slowly cleared from the liver (the residual concentration in the liver tissue was 50–60 ng/g 8 hours after administration). At all time points, the concentrations in the heart (<20 ng/g), lungs (<30 ng/g), spleen (<40 ng/g), and kidneys (<35 ng/g) were extremely low. [1] 3. Metabolism and Excretion: BAMEA-O16B contains bioreducible disulfide bonds, which break in the intracellular reducing environment (high concentration of glutathione), thereby releasing the encapsulated mRNA. The degraded lipid fragments are metabolized in the liver via fatty acid metabolism pathways. 24 hours after administration, approximately 15-20% of the total lipid-derived radioactivity (from ¹⁴C-labeled BAMEA-O16B) was excreted in feces and approximately 5-8% in urine, indicating that feces were the primary route of excretion. [1] |
| Toxicity/Toxicokinetics |
1. In vitro toxicity: In HEK293T, HepG2 and Huh7 cells, concentrations of up to 10 μg/mL of BAMEA-O16B LNPs did not show significant apoptosis induction (detected by Annexin V-FITC/PI double staining; apoptosis rate <5%, while the apoptosis rate of the same concentration of DOTAP LNPs was >15%). No significant disruption of cell membrane integrity was observed (as determined by LDH release assay; LDH release rate <10%). [1] 2. In vivo acute toxicity: C57BL/6 mice were treated with BAMEA-O16B LNPs at doses of 0.25, 0.5, 1.0 and 2.0 mg/kg mRNA. No mouse deaths were observed within 14 days after administration. Mice in all dose groups exhibited normal activity levels, food intake, and water consumption, with no significant decrease in body weight (<5% change in body weight compared to the control group). [1]
3. Organ toxicity: Serum biochemical analysis showed that on days 7 and 14 after administration, the ALT (≤50 U/L), AST (≤120 U/L), BUN (≤8 mmol/L), and creatinine (≤60 μmol/L) levels in mice treated with BAMEA-O16B (0.5 mg/kg mRNA) were within the normal physiological range (same as the saline control group). HE staining of liver, kidney, heart, lung, and spleen tissues showed no signs of inflammation, necrosis, or fibrosis. [1] 4. Immune response: The levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) in the serum of mice treated with BAMEA-O16B (0.5 mg/kg mRNA) at 24 and 72 hours after administration were detected by ELISA. Compared with the control group, no significant increase in cytokine levels was observed (TNF-α <10 pg/mL, IL-6 <15 pg/mL, IL-1β <5 pg/mL), indicating that BAMEA-O16B LNPs do not induce a strong systemic immune response. [1] |
| References | |
| Additional Infomation |
1. Structural features: BAMEA-O16B is a bioreducible cationic lipid with a chemical structure consisting of a central amine core, two C16 alkyl chains (for membrane integration) and disulfide bonds (for intracellular reduction and lipid nanoparticle dissociation). This structure enables it to efficiently encapsulate negatively charged mRNA through electrostatic interactions and release mRNA in a reducing intracellular environment, thereby improving transfection efficiency. [1] 2. Comparison with other lipids: Compared with clinically used lipids (e.g., DLin-MC3-DMA) and conventional cationic lipids (e.g., DOTAP), BAMEA-O16B exhibits higher in vitro and in vivo gene editing efficiency (insertion/deletion frequency is about 2-3 times higher than DOTAP) and lower toxicity (cell viability is about 1.5 times higher than DLin-MC3-DMA at the same mRNA dose), making it an ideal carrier for CRISPR/Cas9-based in vivo gene therapy. [1]
3. Application Potential: BAMEA-O16B-based lipid nanoparticles (LNPs) possess high liver tropism and efficient gene editing capabilities, thus showing potential application value in the treatment of liver-related genetic diseases (e.g., treating familial hypercholesterolemia through Pcsk9 gene editing, and treating hemophilia through factor VIII/IX gene editing). [1] |
| Molecular Formula |
C56H111N3O6S6
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|---|---|
| Molecular Weight |
1114.89
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| Exact Mass |
1113.679
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| CAS # |
2490668-30-7
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| PubChem CID |
155374948
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| Appearance |
Colorless to light yellow liquid
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| LogP |
18.1
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
15
|
| Rotatable Bond Count |
63
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| Heavy Atom Count |
71
|
| Complexity |
1070
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
WBCDKXLTOZQTMM-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C56H111N3O6S6/c1-5-8-11-14-17-20-23-26-29-32-48-66-69-51-45-63-54(60)35-38-57-39-42-58(4)43-44-59(40-36-55(61)64-46-52-70-67-49-33-30-27-24-21-18-15-12-9-6-2)41-37-56(62)65-47-53-71-68-50-34-31-28-25-22-19-16-13-10-7-3/h57H,5-53H2,1-4H3
|
| Chemical Name |
2-(dodecyldisulfanyl)ethyl 3-[2-[2-[bis[3-[2-(dodecyldisulfanyl)ethoxy]-3-oxopropyl]amino]ethyl-methylamino]ethylamino]propanoate
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| Synonyms |
BAMEA-O16B
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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) |
Ethanol : ~100 mg/mL (~89.69 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (2.24 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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 (2.24 mM) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH 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 (2.24 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.8969 mL | 4.4847 mL | 8.9695 mL | |
| 5 mM | 0.1794 mL | 0.8969 mL | 1.7939 mL | |
| 10 mM | 0.0897 mL | 0.4485 mL | 0.8969 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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