Integrins (alphavbeta3, alphavbeta5, alpha5beta1, alphaIIbbeta3). GRGDSPC is an RGD-containing peptide that binds competitively to integrin receptors, which are heterodimeric transmembrane proteins mediating cell adhesion to the extracellular matrix (ECM). The tripeptide motif Arg-Gly-Asp (RGD) is a minimal recognition sequence for several integrins, including alphavbeta3 (vitronectin receptor), alphavbeta5, alpha5beta1 (fibronectin receptor), and alphaIIbbeta3 (fibrinogen receptor on platelets). GRGDSPC binds to the integrin's ligand-binding pocket, competing with native ECM proteins such as fibronectin, vitronectin, fibrinogen, and osteopontin. This binding blocks integrin-mediated cell adhesion, spreading, migration, and survival. The C-terminal cysteine residue provides a free thiol group for bioconjugation (e.g., for immobilizing the peptide on surfaces or coupling to fluorophores or carrier proteins). The peptide is not a drug but a research tool.

In order to control how human mesenchymal stem cells (hMSCs) interact with photocrosslinkable hydrogels, GRGDSPC is conjugated to acrylated dextran via the thiol-acrylate reaction. The conjugation kinetics and efficiency of the GRGDSPC peptide to DEX-MAES16 are assessed by conjugating the peptide to the acrylated Dextran (DEX) macromer at different concentrations (5, 10, and 20 mg/1 g) in PBS at pH 7.8 for varying durations of time (0.25, 0.5, 1 and 3 hours), and quantifying the free thiol groups of unreacted peptides using Ellman's method. Furthermore, the thiol-peptide's reaction kinetics to methacrylated (DEX-HEMA16) and acrylated (DEX-MAES16) macromers are contrasted. The conjugation efficiencies of peptides with DEX-MAES are 105.40, 94.10, and 87.45%, respectively, at as early as 15 minutes of conjugation with 5, 10 and 20 mg of GRGDSPC peptide/1 test. g modified DEX; in contrast, the conjugation efficiencies with DEX-HEMA are 0.73, 15.78, and 18.42%, respectively. Only 35.66% of the peptide's thiol groups react with DEX-HEMA when the GRGDSPC conjugation with DEX-MAES is finished after one hour at a peptide concentration of 10 mg. The kinetics of the reaction are also observed after three hours of conjugation. At this stage, the entire 20 mg GRGDSPC peptide reacts with acrylated DEX, but only 32.53% of the methacrylated DEX responds[1].

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In vitro, GRGDSPC TFA inhibits cell adhesion to RGD-dependent ECM proteins such as fibronectin, vitronectin, and fibrinogen. At concentrations of 50-500 uM, the peptide prevents the attachment of various cell types (e.g., fibroblasts, endothelial cells, platelets, osteoclasts, cancer cells) to ECM-coated surfaces. The inhibitory effect is competitive and reversible. For example, at 250 uM, GRGDSPC significantly reduces the adhesion of wild-type cells to fibronectin. A control peptide with a scrambled sequence (e.g., GRGESP) is used as a negative control and has no effect on adhesion. GRGDSPC also inhibits cell spreading, migration (in wound-healing and Transwell assays), and integrin-mediated signaling (e.g., FAK, Src, PI3K/AKT) in a dose-dependent manner (IC50 typically 50-200 uM). The peptide's potency varies depending on the integrin subtype and the cell type. The presence of the cysteine residue does not affect RGD-binding activity but enables peptide immobilization. In studies of mineralization (bone formation), GRGDSPC (0.1-500 uM) inhibits osteoblast-mediated mineralization in fetal rat parietal bone cultures, as measured by calcium content and bone area. The peptide also inhibits platelet aggregation and clot retraction in vitro by blocking alphaIIbbeta3 integrins. Additionally, GRGDSPC can be used as a surface coating to promote cell adhesion when immobilized, but in soluble form, it acts as an inhibitor.

Not applicable. The primary use of GRGDSPC TFA is as an in vitro research tool; it is not administered in vivo as a therapeutic agent. However, the peptide can be used in animal models when delivered locally (e.g., intratumoral injection) or systemically (e.g., intravenous) to block integrin function. For example, in rat models of hepatocellular carcinoma, transarterial infusion of GRGDSP (without cysteine) loaded in nanoparticles has been shown to reduce tumor growth and metastasis by inhibiting integrin-mediated tumor cell adhesion and angiogenesis. The presence of cysteine allows for conjugation to nanoparticles or biomaterials for targeted delivery. In vivo, soluble RGD peptides have a short plasma half-life (minutes) due to rapid renal clearance and proteolytic degradation. Thus, for sustained in vivo effects, the peptide is often conjugated to carriers (e.g., PEG, nanoparticles) or administered locally. No approved in vivo indications exist for GRGDSPC; it is strictly a research reagent.

To assess integrin-binding affinity and specificity, a solid-phase binding assay or surface plasmon resonance (SPR) is used. For a direct binding ELISA: 96-well MaxiSorp plates are coated with purified integrin (e.g., alphavbeta3, alpha5beta1, or alphaIIbbeta3) at 1-5 ug/mL in carbonate-bicarbonate buffer (pH 9.6) overnight at 4degC. Plates are blocked with 1% BSA in PBS for 1 hour at 37degC. Biotinylated GRGDSPC (biotin-GRGDSPC) is prepared (the cysteine provides a site for biotinylation, but biotin is typically added via the N-terminus or Lys side chain). Varying concentrations of biotin-GRGDSPC (0.01-1000 uM) are added and incubated for 2 hours at room temperature. After washing, HRP-conjugated streptavidin is added, followed by TMB substrate. Absorbance is read at 450 nm. The Kd can be calculated from binding curves. For competitive binding assays, plates are coated with ECM protein (e.g., fibronectin, 5-10 ug/mL). Cells (e.g., 5 × 10^4/well) are pre-incubated with increasing concentrations of GRGDSPC (0-1000 uM) for 20 min at 37degC, then added to coated wells. After 30-60 min, non-adherent cells are washed off, and adherent cells are fixed, stained with crystal violet, and dissolved in 1% SDS; absorbance is read at 590 nm. The IC50 is determined. For SPR: Integrin protein is immobilized on a CM5 sensor chip. GRGDSPC in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MnCl2 (to activate integrin), 0.005% P20) is flowed over at concentrations 0.1-1000 uM. The sensorgram is recorded, and KD is calculated by steady-state analysis. The peptide is also tested for competitive displacement of a known RGD ligand.

For cell adhesion assays, any adherent cell line expressing relevant integrins (e.g., HEK293, NIH3T3, HUVECs, MDA-MB-231, platelets) can be used. Cells are cultured in appropriate medium (DMEM, RPMI-1640, etc.) with 10% FBS. On the day of the assay, 96-well plates are coated with ECM proteins (e.g., fibronectin (5-10 ug/mL), vitronectin (2-5 ug/mL), or fibrinogen (10-20 ug/mL)) in PBS overnight at 4degC, then blocked with 1% BSA (or 1% heat-denatured BSA) in PBS for 1 hour at 37degC. Cells are detached with trypsin-EDTA, washed with serum-free medium, and resuspended in serum-free medium. Cells are pre-incubated with GRGDSPC TFA (0-1000 uM) for 15-30 minutes at 37degC with gentle agitation. Control cells are treated with vehicle (water or PBS) or a control peptide (e.g., GRGESP). The cell suspension (50-100 uL, 5 × 10^4 cells/well) is added to the coated wells and allowed to adhere for 30-60 minutes at 37degC. Non-adherent cells are removed by gently washing the wells 2-3 times with PBS. Adherent cells are fixed with 4% paraformaldehyde (10 min), stained with 0.1% crystal violet (15 min), washed, and solubilized with 1% SDS (100 uL/well). Absorbance is measured at 590 nm using a microplate reader. Percent inhibition = (1 - (A590 (treated) / A590 (control))) × 100. IC50 values (typically 50-200 uM) are calculated by fitting a four-parameter logistic curve. For cell spreading assays: After adhesion for 15-60 min, cells are fixed, stained with phalloidin (F-actin) and DAPI, and cell spreading (cell area, circularity) is quantified by fluorescence microscopy. For migration assays: In Transwell chambers (8-um pores), lower chamber contains 10% FBS as chemoattractant, cells in the upper chamber (serum-free medium) are pre-incubated with GRGDSPC (0-500 uM) for 30 min. After 4-24 hours, migrated cells are fixed, stained with crystal violet, and counted. For scratch-wound healing assays: A confluent cell monolayer is scratched with a pipette tip, and cells are treated with GRGDSPC (0-500 uM) in medium with 1% FBS. Wound closure is monitored by microscopy at 0, 6, 12, 24 hours and quantified by ImageJ. The cysteine residue in GRGDSPC does not interfere with cell adhesion assays unless reduced DTT is added (which may break disulfide bonds). The TFA salt is soluble in water and PBS. For surface immobilization applications: GRGDSPC can be coupled to maleimide-activated surfaces (e.g., gold, glass, nanoparticles) via the cysteine thiol group. After immobilization, the peptide promotes cell adhesion rather than inhibiting it. In such assays, surfaces are incubated with GRGDSPC (0.1-1 mM in PBS, pH 7.4) for 2-4 hours, washed, and then cells are plated. Cell adhesion to immobilized GRGDSPC is measured. All experiments should be performed in triplicate, with at least three independent experiments.

Not applicable for soluble use. For in vivo studies, GRGDSPC can be used as a control peptide to block integrin function in animal models, but it has poor PK properties (short half-life). A typical protocol for evaluating the in vivo anti-adhesive effects of a GRGDSPC conjugate (e.g., GRGDSPC immobilized on nanoparticles) uses a rat model of hepatocellular carcinoma. Rats (male, 200-250 g) bearing orthotopic liver tumors are prepared. The peptide conjugate (e.g., GRGDSPC-conjugated nanoparticles loaded with doxorubicin) is administered by transarterial infusion (via the hepatic artery) at a dose of 0.5-2 mg peptide/kg. The compound is injected over 5-10 minutes. Control groups receive free peptide, control peptide (GRGESP), or vehicle. Tumor growth is monitored by MRI or ultrasound over 2-4 weeks. At sacrifice, tumors are excised, weighed, and analyzed for histology (necrosis, apoptosis), microvessel density (CD31 staining), and integrin expression. For systemic administration (e.g., intravenous injection) to block platelet aggregation or cancer cell metastasis, GRGDSPC (10-50 mg/kg) is injected into the tail vein of mice. The compound is rapidly cleared, so multiple doses or infusion may be required. No standard in vivo protocol is available for GRGDSPC alone; it is typically conjugated. All animal procedures must be approved by IACUC.

Not applicable. GRGDSPC TFA is not a drug candidate, and no pharmacokinetic data are available for this specific peptide. As a 7-amino acid peptide (MW ~780 g/mol), GRGDSPC is highly water-soluble and will be rapidly cleared from the circulation via renal filtration (glomerular filtration) and proteolytic degradation in plasma (t1/2 < 5 min). The peptide is not orally bioavailable. The TFA salt does not alter its PK properties. For in vivo applications, GRGDSPC is typically conjugated to a polymer (PEG), nanoparticle, or carrier protein to extend circulation time and enhance targeting. The conjugated forms have longer half-lives (hours) depending on the size of the carrier. The parent free peptide is not used for pharmacokinetic analysis. GRGDSPC is for research use only and is not intended for human or animal therapy.

No specific toxicity data are available for GRGDSPC TFA. As a short synthetic peptide composed of naturally occurring L-amino acids, GRGDSPC is generally considered to have low toxicity. In vitro, the peptide is not cytotoxic to cells at concentrations up to 1 mM, as assessed by MTT or LDH release assays. In vivo, free GRGDSPC administered intravenously at doses up to 50 mg/kg in mice is generally well-tolerated, with no acute toxicity or mortality reported in the literature. However, high doses may cause transient hypotension and bleeding due to inhibition of platelet aggregation via alphaIIbbeta3 integrin blockade, but this is a pharmacological effect, not toxicity. The TFA salt is present in small amounts and is not toxic. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been conducted. The peptide is for research use only and is not approved for human therapy. Standard laboratory safety precautions (gloves, lab coat) are sufficient. The peptide is not intended for in vivo use without proper formulation and carrier.

[1]. Nguyen MK, et al. Photocrosslinkable, biodegradable hydrogels with controlled cell adhesivity for prolonged siRNAdelivery to hMSCs to enhance their osteogenic differentiation. J Mater Chem B. 2017 Jan 21;5(3):485-495.

The RGD (Arg-Gly-Asp) motif is the most well-known and widely studied cell adhesion sequence, present in many ECM proteins including fibronectin, vitronectin, fibrinogen, laminin, and collagen. RGD peptides are used extensively in cell biology, biomaterials, tissue engineering, and cancer research. GRGDSPC is a variant of the GRGDSP peptide (which was originally identified as an inhibitor of integrin-fibronectin binding) with an extra cysteine residue at the C-terminus. The cysteine allows for site-specific conjugation to maleimide-activated surfaces or molecules, enabling the creation of RGD-functionalized biomaterials (e.g., hydrogels, scaffolds, nanoparticles) for studies of cell-material interactions. The peptide is also used as a competitive inhibitor of integrin binding in solution. The control peptide GRGESP (where Asp is replaced by Glu) or GRADSP is used as a negative control. The TFA salt is used to improve solubility and stability. GRGDSPC is not a drug and has not received regulatory approval for any therapeutic indication. It is for research use only.

C27H43F3N10O13S

804.75

GRGDSPC;91575-26-7

White to off-white solid powder

2934.99.9001

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 (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :≥ 100 mg/mL (~124.26 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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