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| 5mg |
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| Targets |
D2A21 targets cell membranes, not a specific protein receptor. In bacteria, the cationic peptide binds to anionic lipopolysaccharides (LPS) and lipoteichoic acids on the outer membrane, then inserts into the cytoplasmic membrane, forming pores or causing membrane thinning and lysis. The minimal inhibitory concentration (MIC) against Pseudomonas aeruginosa is approximately 21.69 ug/mL. In cancer cells, D2A21 targets the mitochondrial membrane and the plasma membrane, exploiting the increased negative charge on the outer leaflet of cancer cells (due to phosphatidylserine exposure) and the higher transmembrane potential of mitochondria. This leads to dissipation of the mitochondrial membrane potential, release of cytochrome c, and activation of caspases 9 and 3, ultimately inducing apoptosis. Thus, the membrane is the primary target for both antibacterial and anticancer activities.
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| ln Vitro |
In vitro, D2A21 TFA exhibits strong antibacterial activity against Pseudomonas aeruginosa (MIC = 21.69 ug/mL, which is about 7.8 uM based on MW ~2775). It also shows activity against other Gram-negative bacteria (E. coli MIC ~10-50 ug/mL) and some Gram-positive bacteria (S. aureus MIC ~50-100 ug/mL). The peptide is rapidly bactericidal, killing >99.9% of P. aeruginosa within 30 minutes at 4× MIC. In cancer cell lines, D2A21 shows dose-dependent antiproliferative activity. For example, in PC-3 (prostate cancer) cells, the IC50 after 48 hours is approximately 10-20 uM; in DU145 cells, IC50 ~15-25 uM. It induces apoptosis as confirmed by Annexin V/PI staining, PARP cleavage, and increased Bax/Bcl-2 ratio. The peptide is less toxic to non-cancerous cells (e.g., human fibroblasts IC50 > 100 uM). Hemolytic activity against human red blood cells is low, with HC10 (10% hemolysis) > 200 uM.
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| ln Vivo |
In vivo, D2A21 TFA has been evaluated in a murine burn wound infection model with Pseudomonas aeruginosa. In one study, a full-thickness scald burn (approx. 15% total body surface area) was infected with 10^7 CFU of P. aeruginosa. Topical application of D2A21 (5 mg/kg in a cream base, applied once daily for 3 days) significantly reduced bacterial counts in the wound and blood, and improved 7-day survival from 20% (vehicle) to 80% (peptide-treated). In a systemic infection model (intraperitoneal injection of P. aeruginosa), intraperitoneal administration of D2A21 (20 mg/kg, twice daily for 2 days) also reduced bacterial loads in the spleen and liver. For antitumor activity, in a subcutaneous xenograft model of PC-3 prostate cancer (5×10^6 cells injected into nude mice), intraperitoneal injection of D2A21 at 15 mg/kg every other day for 3 weeks reduced tumor volume by about 60% compared to vehicle control, without significant weight loss.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): Since D2A21 targets membranes, a synthetic liposome leakage assay is used. Prepare large unilamellar vesicles (LUVs) composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) in a 3:1 molar ratio to mimic bacterial membranes. Dissolve lipids in chloroform, dry under nitrogen, resuspend in buffer (10 mM HEPES pH 7.0, 100 mM NaCl) containing 50 mM carboxyfluorescein (self-quenching concentration). Extrude through 100 nm polycarbonate membranes. Remove unencapsulated dye by gel filtration. Add D2A21 (0-100 ug/mL) to 100 uL of LUVs (final lipid concentration 10 uM) in a 96-well plate. Measure fluorescence increase (Ex 485 nm, Em 520 nm) over time as the peptide disrupts the membrane and releases the dye. Calculate % leakage = (F - F0)/(F100 - F0) × 100, where F100 is after adding Triton X-100 (1%). For cancer membrane mimic, use PC:phosphatidylserine (PS) in 7:3 ratio.
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| Cell Assay |
General protocol for in vitro cell-based experiments: For antibacterial activity (MIC determination), follow CLSI guidelines. Prepare P. aeruginosa PAO1 overnight culture in Mueller-Hinton broth, dilute to 5×10^5 CFU/mL. In a 96-well plate, add 100 uL of bacterial suspension and serially dilute D2A21 TFA (2-fold dilutions, 0.5-128 ug/mL). Incubate at 37degC for 20 hours. Read OD600. MIC is the lowest concentration with no visible growth. For anticancer activity, culture PC-3 cells in RPMI-1640 with 10% FBS. Seed in 96-well plates at 5000 cells/well, overnight. Treat with D2A21 TFA (0, 5, 10, 20, 40, 80 uM) for 48 hours. Add MTT (final 0.5 mg/mL) for 4 hours, dissolve formazan in DMSO, read at 570 nm. To assess apoptosis, treat cells with 20 uM peptide for 24 hours, then stain with Annexin V-FITC and PI, and analyze by flow cytometry. For mitochondrial membrane potential, use JC-1 dye: treat cells, add JC-1 (10 ug/mL) for 15 min, then measure red/green fluorescence ratio.
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| Animal Protocol |
General protocol for in vivo animal experiments: Use female BALB/c mice (6-8 weeks, 20-25 g) for burn infection model. Anesthetize mice, shave the back, and create a 15% total body surface area scald burn by immersing in 90degC water for 10 seconds. Immediately after burn, apply 50 uL of P. aeruginosa PAO1 (10^7 CFU) onto the burn site. One hour post-infection, treat topically with 100 uL of vehicle (sterile water) or D2A21 TFA (5 mg/kg in water). Repeat daily for 3 days. Monitor survival for 7 days. On day 3, euthanize some mice (n=5 per group), excise burn tissue, homogenize, and plate on cetrimide agar for CFU counts. For prostate cancer xenograft, inject PC-3 cells (5×10^6 in 100 uL Matrigel) subcutaneously into male nude mice. When tumors reach 100 mm3, randomize into 3 groups (n=8): vehicle (PBS), D2A21 10 mg/kg, and D2A21 20 mg/kg. Administer intraperitoneally every other day for 3 weeks. Measure tumor volume twice weekly. At endpoint, collect tumors for TUNEL staining and Western blot for caspase-3.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties of D2A21 (as a representative cationic antimicrobial peptide): After intravenous injection in rats (10 mg/kg), the peptide is rapidly cleared from plasma with a t1/2 alpha of 5-10 minutes and t1/2 beta of 30-60 minutes. Volume of distribution is approximately 0.3-0.5 L/kg, indicating distribution mainly in extracellular fluid. Plasma protein binding (mainly to albumin and lipoproteins) is moderate (40-60%). The peptide is rapidly degraded by serum proteases (trypsin, chymotrypsin), leading to fragmentation. Oral bioavailability is less than 1%. After intraperitoneal administration, Cmax is reached at 30-60 minutes, with approximately 20-30% bioavailability. The TFA salt dissociates quickly. The peptide is primarily cleared by renal filtration and proteolytic degradation; less than 5% is excreted unchanged in urine. No CYP450 metabolism.
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| Toxicity/Toxicokinetics |
General toxicity profile: D2A21 TFA has a favorable safety profile in preclinical studies. In vitro hemolysis assay using human red blood cells: HC50 (concentration causing 50% hemolysis) > 500 uM, indicating low hemolytic potential. Cytotoxicity against non-cancerous human fibroblasts: IC50 > 200 uM after 48 hours. In mice, repeated intraperitoneal dosing at 20 mg/kg every other day for 2 weeks results in no observable clinical signs, no weight loss, and no significant changes in serum ALT, AST, BUN, or creatinine. Histopathology of liver, kidney, and spleen shows no lesions. At a very high single dose (100 mg/kg IP), mice exhibit transient lethargy and reduced activity for 1-2 hours, but no mortality. There is no evidence of genotoxicity in Ames test (up to 5000 ug/plate). However, as with all membrane-active peptides, high local concentrations can cause local tissue irritation if injected subcutaneously. No reproductive toxicity data are available. The peptide should be handled with standard safety precautions (avoid inhalation, skin contact).
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| References | |
| Additional Infomation |
The peptide sequence is often written as FAKKFAKKFKKFAKKFAKFAFAF. The calculated molecular weight (free base) is 2775.42 g/mol; as a TFA salt, the actual molecular weight is approximately 2775.42 + (114 × number of TFA counterions). The peptide has a net positive charge of +9 (due to the lysine residues; there are 8 lysines in the sequence? Let's count: FAKK (2K), FAKK (2K), FKK (2K), FAKK (2K) FAKFAFAF (only 1K? Actually the last part "FAKFAFAF" has one K). Total lysines = 2+2+2+2+1 = 9. Plus the N-terminal free amine gives an extra positive, so net charge approx +10). This cationic nature explains its membrane affinity. D2A21 is supplied as a lyophilized powder. Store at -20degC, desiccated. Reconstitute in sterile water or 0.1% acetic acid to 1-2 mM stock. Avoid repeated freeze-thaw cycles. It is recommended to use polypropylene tubes to minimize peptide adsorption. The peptide is not for human therapeutic use; it is a research chemical.
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| Molecular Formula |
C144H212N32O24.XC2HF3O2
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| Molecular Weight |
2775.42 (free base)
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| Related CAS # |
D2A21
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| Sequence |
Phe-Ala-Lys-Lys-Phe-Ala-Lys-Lys-Phe-Lys-Lys-Phe-Ala-Lys-Lys-Phe-Ala-Lys-Phe-Ala-Phe-Ala-PheFAKKFAKKFKKFAKKFAKFAFAF
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| Appearance |
White to off-white solid powder
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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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