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| Targets |
CD13
The primary target of the NGR peptide is a specific isoform of aminopeptidase N (APN/CD13), a zinc-dependent metalloprotease. This isoform is highly expressed on the surface of endothelial cells in tumor blood vessels but is not present on normal, quiescent endothelium. The binding of the NGR motif to CD13 is conformation-dependent, with the cyclic form (CNGRCG) exhibiting significantly higher binding affinity and stability than the linear form. Upon binding, the NGR peptide can be internalized via receptor-mediated endocytosis, making it an efficient vehicle for intracellular drug delivery. The selectivity for the tumor vasculature is a key feature for targeted cancer therapies, allowing for enhanced drug accumulation at the tumor site while reducing systemic toxicity. |
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| ln Vitro |
In vitro, the NGR peptide itself is not directly cytotoxic but serves as a delivery vehicle. Its activity is measured by its binding affinity to CD13-positive cells and its ability to enhance the uptake of conjugated drugs. For instance, cyclic NGR peptide-daunomycin conjugates demonstrate significant cytotoxic effects against CD13-positive tumor cells (HT-1080 fibrosarcoma) while maintaining lower toxicity towards CD13-negative cells (HT-29 colon cancer). NGR peptide-TNF (tumor necrosis factor) fusion proteins have shown increased anti-tumor activity by disrupting the tumor vasculature. The selectivity ratio is often high, providing a large therapeutic index. The peptide is a ligand with high specificity and high affinity for CD13. The cyclic NGR peptide is a strong and specific binder.
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| ln Vivo |
One possible SPECT reagent for tumor imaging and early diagnosis is NGR peptide trifluoroacetate (20 mg/kg; IV) [2].
In vivo, the NGR peptide has been used extensively in animal models to demonstrate targeted drug delivery. In the HepG2 mouse tumor model, intravenous administration of ⁹⁹ᵐTc-labeled NGR peptide (20 mg/kg) resulted in high tumor uptake, which could be blocked by co-injection with an excess of unlabeled NGR peptide, confirming specificity. Doxorubicin (DOX) coupled to an NGR peptide displays enhanced anti-tumor effects with even lower toxicity than the free drug. In mice, the peptide has also been used to create NGR-modified hydrogels for sustained release at the tumor site. The peptide's ability to recognize CD13 on tumor vessels allows for effective vascular targeting, making it a versatile tool for both imaging and therapy. |
| Enzyme Assay |
A cell-free receptor binding assay can be performed using recombinant CD13/APN protein. A 96-well plate is coated with 5 ug/mL of human recombinant CD13/APN in carbonate-bicarbonate buffer (pH 9.6) overnight at 4degC. The plate is then blocked with 5% BSA in PBS for 2 hours at 37degC. Serial dilutions of the NGR peptide (0.1-1000 nM) are added to the wells and incubated for 2 hours at 37degC. After washing, a biotinylated anti-NGR antibody or a labeled form of the NGR peptide (e.g., NGR-FITC) is added to detect binding. For a competition assay, a fixed concentration of biotinylated NGR (10 nM) is mixed with increasing concentrations of unlabeled NGR peptide. After a second incubation and washing steps, HRP-conjugated streptavidin is added, followed by TMB substrate. The reaction is stopped with H2SO4, and the absorbance is read at 450 nm. The binding affinity (Kd) and the IC50 for competition are determined. The cyclic NGR peptide shows a Kd in the low nanomolar range, demonstrating high affinity.
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| Cell Assay |
A CD13-positive cell line, such as HT-1080 fibrosarcoma cells, is used for cell-based assays. For a flow cytometry binding assay, 5×10⁵ HT-1080 cells are harvested and resuspended in FACS buffer (PBS + 1% BSA). The cells are incubated with varying concentrations of a fluorescently labeled NGR peptide (e.g., NGR-FITC, 0.1-1000 nM) on ice for 30 minutes in the dark. After incubation, the cells are washed three times with ice-cold FACS buffer to remove unbound peptide. The cells are then resuspended in 300 uL of FACS buffer and analyzed using a flow cytometer (e.g., BD FACSCalibur). For blocking studies, cells are pre-incubated with a 100-fold excess of unlabeled NGR peptide or an anti-CD13 antibody for 30 minutes before adding the labeled peptide. The mean fluorescence intensity (MFI) is used to quantify the binding. A shift in MFI indicates binding, and a reduction in MFI in the blocking groups confirms specificity. The concentration at which 50% of the receptors are bound is the apparent Kd.
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| Animal Protocol |
Animal/Disease Models: 4-6 weeks, BALB/c nude mice (HepG2 tumor model)[2]
Doses: 20 mg/kg Route of Administration: Iv Experimental Results: Blocked the tumor uptake for 99mTc-NGR. The in vivo targeting ability of NGR peptide conjugates can be assessed in a subcutaneous xenograft mouse model. Female BALB/c nude mice (4-6 weeks old, ~20 g) are injected subcutaneously with 5×10⁶ HT-1080 (CD13-positive) tumor cells in the right flank. When the tumor volume reaches 150-250 mm3, the mice are randomly divided into groups (n=5 per group). For an imaging study, the mice are injected intravenously (tail vein) with ⁶⁸Ga-labeled NGR peptide (5-10 MBq, ~10 ug). PET/CT scans are performed at 30, 60, and 120 minutes post-injection. Regions of interest (ROIs) are drawn over the tumor and major organs to calculate the percentage of injected dose per gram (%ID/g) and tumor-to-muscle ratios. For a blocking study, a separate group of mice is co-injected with an excess (100 ug) of unlabeled NGR peptide 30 minutes prior to the injection of the labeled peptide. A significant reduction in tumor uptake in the blocking group (>70% reduction) confirms the CD13-mediated specificity of the probe. For a therapy study, NGR-DOX conjugates (e.g., 5 mg/kg DOX equivalent) are injected intravenously on days 1, 4, 7, and 10. Tumor volumes are measured with calipers for 4-6 weeks. The NGR-DOX group should show significantly slower tumor growth and lower systemic toxicity (measured by body weight loss) compared to the free DOX group. |
| ADME/Pharmacokinetics |
The NGR peptide Trifluoroacetate has a molecular formula of C22H37F3N10O10S2 and a molecular weight of 722.72 g/mol. The peptide has a cyclic structure (CNGRCG). The lyophilized powder should be stored at -20degC in a sealed container, protected from light and moisture, where it is stable for up to 3 years. The TFA salt form is used to enhance its water solubility and stability. It is readily soluble in water and DMSO. For in vivo use, the peptide can be formulated in sterile saline or PBS. The cyclic structure is essential for maintaining high receptor binding affinity. The peptide is relatively stable in serum but may be subject to proteolytic degradation over time, which is a consideration for in vivo experiments. Its small size (722 Da) allows for rapid tissue penetration and clearance, mainly via renal filtration, leading to high tumor-to-background ratios at early time points.
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| Toxicity/Toxicokinetics |
The NGR peptide is for research use only and is not for clinical use. No specific toxicity data is available for the peptide itself. However, toxicological data is available for the therapeutic conjugate NGR-hTNF, which has been in clinical trials. Grade 1-2 chills (58%) and fever (56%) were the most common toxicities observed. Subcutaneous administration at high doses was associated with thrombo-embolic events, although these were not reported with intravenous administration. Standard laboratory safety precautions, including the use of PPE, should be followed when handling the compound. The TFA counterion can be toxic if ingested or inhaled in large quantities. The product is not a drug and has not received FDA approval.
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| References | |
| Additional Infomation |
The NGR peptide is a powerful tool for tumor vascular targeting and has been widely used to deliver a variety of payloads, including chemotherapy (Doxorubicin), pro-apoptotic peptides (KLAKLAKKLAKLAK), tumor necrosis factor alpha (TNF-alpha), and imaging agents (99mTc, 68Ga, Cy5.5). The cyclic NGR peptide shows superior antitumor activity and stability compared to its linear counterpart. It can be combined with other targeting motifs like RGD (Arg-Gly-Asp) to create dual-targeting conjugates for tumors with heterogeneous receptor expression. The peptide can also be integrated into hydrogels (e.g., NGR-GelMA) for localized drug delivery. The TFA salt is the standard research form of this synthetic peptide. It is an essential tool for developing new targeted therapies and diagnostics for cancer. It is also used for basic research on the CD13 receptor and its role in angiogenesis. The cyclic NGR peptide specifically binds to CD13 with the highest selectivity. It is a pure research product not intended for human use.
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| Molecular Formula |
C22H37F3N10O10S2
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| Molecular Weight |
722.72
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| Related CAS # |
NGR peptide;651328-78-8
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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 |
| 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) |
H2O :~50 mg/mL (~69.18 mM)
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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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3837 mL | 6.9183 mL | 13.8366 mL | |
| 5 mM | 0.2767 mL | 1.3837 mL | 2.7673 mL | |
| 10 mM | 0.1384 mL | 0.6918 mL | 1.3837 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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