Integrin[1].
Integrins (alphavbeta3, alphavbeta5, alpha5beta1, alphaIIbbeta3). GRGDSP is an RGD-containing peptide that binds to the ligand-binding pocket of integrin receptors, which are heterodimeric transmembrane proteins that mediate cell-ECM adhesion. Integrins recognize the RGD motif present in many ECM proteins, including fibronectin, vitronectin, fibrinogen, osteopontin, and tenascin. GRGDSP competes with these native proteins for integrin binding, thereby inhibiting integrin-dependent cell adhesion to the ECM. The inhibition is competitive, reversible, and specific for RGD-binding integrins. The peptide does not affect integrins that recognize other motifs (e.g., alpha4beta1 binding to VCAM-1 or alpha2beta1 binding to collagen). The control peptide GRGESP (where Asp is replaced by Glu) or GRADSP does not inhibit integrin binding and serves as a negative control. GRGDSP is not a drug but a widely used research reagent.

Transarterial GRGDSP infusion has been shown to be effective (Gly-Arg-Gly-Asp-Ser-Pro integrin-inhibitor which includes RGD-peptide). As a synthesized linear RGD peptide, GRGDSP(Gly-Arg-Gly-Asp-Ser-Pro) can prevent tumor cells from adhering to blood vessel endothelial cells and restrict their ability to spread[1]. A soluble integrin-blocking RGD-based peptide called GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) is employed. In integrin research, GRGDSP is frequently utilized in conjunction with other RGD peptides. In order to encourage endothelialization, GRGDSP can be applied to the surface of cardiovascular implants, such as vascular grafts[2].

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In vitro, GRGDSP TFA (Gly-Arg-Gly-Asp-Ser-Pro) is a potent and specific inhibitor of integrin-mediated cell adhesion. At concentrations of 50-500 uM, GRGDSP significantly inhibits the attachment of various cell types (e.g., fibroblasts, epithelial cells, endothelial cells, osteoclasts, platelets, and cancer cells) to RGD-dependent ECM proteins, including fibronectin, vitronectin, and fibrinogen. For example, at 100-500 uM, GRGDSP reduces cell adhesion to fibronectin-coated surfaces by 50-90% as measured by crystal violet staining. The IC50 for inhibiting adhesion to fibronectin is typically 50-200 uM. GRGDSP also inhibits cell spreading on ECM substrates, as assessed by phalloidin staining of F-actin, and prevents the formation of focal adhesions (reduced paxillin or vinculin staining). In migration assays, GRGDSP (50-500 uM) inhibits wound-healing closure and Transwell migration in a dose-dependent manner. In platelet aggregation assays, GRGDSP (0.1-1 mM) blocks fibrinogen binding to alphaIIbbeta3 integrin, thereby inhibiting platelet aggregation and clot formation. In angiogenesis assays, GRGDSP inhibits endothelial cell tube formation on Matrigel. GRGDSP also inhibits integrin-mediated signaling, including FAK (Tyr397) phosphorylation, Src activation, and downstream PI3K/AKT and ERK1/2 pathways. The peptide does not inhibit integrin binding to non-RGD ligands (e.g., laminin, collagen). The control peptide GRGESP has no inhibitory activity. The TFA salt does not affect activity. In studies of bone formation, GRGDSP (100-500 uM) inhibits mineralization in fetal rat parietal bone cultures.

Not applicable (soluble integrin inhibitor). The primary use of GRGDSP TFA is as an in vitro research tool to block integrin function. However, the peptide can be used in vivo to study integrin-mediated processes in animal models when administered locally or systemically. For example, in rat models of hepatocellular carcinoma, transarterial infusion of GRGDSP (loaded in nanoparticles) reduces tumor growth and metastasis by inhibiting integrin-mediated tumor cell adhesion and angiogenesis. In chicken chorioallantoic membrane (CAM) assays, GRGDSP (50-200 uM) inhibits angiogenesis. In vivo, soluble RGD peptides have a very short plasma half-life (minutes) due to renal clearance and proteolytic degradation; thus, they are often conjugated to carriers (e.g., PEG, nanoparticles) for sustained effect. No approved in vivo therapeutic indications exist for GRGDSP; it is strictly a research reagent. The peptide can also be used ex vivo: tissue sections can be incubated with GRGDSP (100-500 uM) prior to adding fluorescently labeled ECM proteins to assess integrin binding.

To assess integrin binding inhibition, a solid-phase competitive adhesion assay is standard. 96-well plates are coated with an ECM protein (e.g., fibronectin (5-10 ug/mL), vitronectin (2-5 ug/mL), or fibrinogen (10-20 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. Cells (e.g., HEK293, NIH3T3, or platelets) are detached with trypsin-EDTA, washed with serum-free medium, and resuspended in serum-free medium. GRGDSP TFA (0-1000 uM) is added to the cell suspension, and cells are pre-incubated for 15-30 minutes at 37degC. The cell suspension (5 × 10^4 cells/well) is added to the ECM-coated wells and incubated for 30-60 minutes at 37degC. Non-adherent cells are removed by gentle washing with PBS. Adherent cells are fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, washed, and lysed with 1% SDS. Absorbance is read at 590 nm. Percent inhibition is calculated relative to untreated cells. The IC50 is derived from a dose-response curve (four-parameter logistic fit). For a direct binding assay, an ELISA can be performed: Coat plate with purified integrin (e.g., alphavbeta3 or alpha5beta1). After blocking, add biotinylated GRGDSP (biotin-GRGDSP, if available) +/- unlabeled GRGDSP, detect with streptavidin-HRP and TMB substrate. For surface plasmon resonance (SPR): Immobilize integrin on a CM5 sensor chip, flow GRGDSP (0.1-1000 uM) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MnCl2, 0.005% P20) at 25degC. Record sensorgrams, and determine KD and binding kinetics.

For cell adhesion assays (detailed in Section 5), any adherent cell line expressing RGD-binding integrins is suitable. Cells are cultured in standard medium. 96-well plates are coated with fibronectin (5-10 ug/mL) in PBS overnight at 4degC, then blocked with 1% BSA (30 min, 37degC). Cells are trypsinized, washed, and resuspended in serum-free medium. GRGDSP TFA (0-1000 uM, prepared as a 100 mM stock in water or PBS, store at -20degC) is added to the cell suspension and pre-incubated for 15-30 min at 37degC. Cells are seeded at 5 × 10^4 cells/well and incubated for 30-60 min at 37degC. Non-adherent cells are removed by washing 2-3 times with PBS. Adherent cells are fixed (4% PFA, 10 min), stained with crystal violet (0.1%, 15 min), washed, and lysed in 1% SDS. Absorbance at 590 nm is measured. Percent adhesion = (OD590 treated / OD590 control) × 100. IC50 is calculated from a dose-response curve (typically 50-200 uM). For cell spreading assays: after adhesion (15-60 min), cells are fixed, permeabilized with 0.1% Triton X-100, stained with phalloidin-FITC (F-actin) and DAPI (nucleus), and examined by fluorescence microscopy. Cell area is quantified using ImageJ. For migration assays: Transwell inserts (8-um pores) are coated with fibronectin (10 ug/mL) on both sides. Cells (5 × 10^4 in 100 uL serum-free medium) pre-treated with GRGDSP (0-500 uM) are added to the upper chamber. The lower chamber contains 10% FBS as chemoattractant. After 6-24 hours, non-migrated cells are removed from the upper surface, and migrated cells on the lower surface are stained with crystal violet and counted (5 fields per insert). For scratch-wound healing assays: Confluent cells in a 6-well plate are scratched with a pipette tip, washed, and incubated in medium containing 1% FBS and GRGDSP (0-500 uM). Wound closure is measured at 0, 6, 12, 24 h using a phase-contrast microscope. For signaling assays: Cells are plated on fibronectin-coated dishes and allowed to attach for 30-60 min in the presence or absence of GRGDSP. Cells are lysed, and cell lysates are analyzed by Western blot for p-FAK (Tyr397), p-Src (Tyr416), p-AKT (Ser473), p-ERK (Thr202/Tyr204), or total proteins. All experiments should be performed in triplicate, with at least three independent experiments. For platelet aggregation assays: Human platelet-rich plasma (PRP) is incubated with GRGDSP (0-1 mM) for 5 min, then aggregation is induced by ADP (10 uM), collagen (2 ug/mL), or thrombin (0.1 U/mL). Aggregation is monitored by light transmission aggregometry. The TFA salt is soluble in water and PBS. Note: GRGDSP is not cytotoxic at these concentrations.

For in vivo studies, soluble GRGDSP is not typically used due to its short half-life, but it can be conjugated to nanoparticles or delivered locally. One example: rat model of hepatocellular carcinoma. Male Sprague-Dawley rats (200-250 g) bearing orthotopic liver tumors are prepared by intrahepatic injection of cancer cells (e.g., CBRH-7919). GRGDSP is formulated as a conjugate: e.g., GRGDSP coupled to poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) nanoparticles (encapsulating doxorubicin). The GRGDSP-conjugated nanoparticles (dose: 0.5-2 mg peptide equivalent/kg in 200 uL saline) are administered by transarterial infusion via the hepatic artery over 5-10 minutes. Control groups: free GRGDSP (same dose), untargeted nanoparticles (no GRGDSP), GRGESP-conjugated nanoparticles, or vehicle (saline). Treatment is given on day 0 and day 7. Tumors are measured by MRI or ultrasound at baseline and day 14. On day 14, rats are euthanized, tumors are excised, weighed, sectioned, and stained with H&E and CD31 (for microvessel density). TUNEL assay is used to assess apoptosis. For systemic administration: Mice are injected intravenously with GRGDSP (10-50 mg/kg in 100-200 uL PBS). Blood samples are collected at various time points to measure inhibition of platelet aggregation (by aggregometry) and to assess anti-metastatic efficacy (e.g., in B16-F10 melanoma lung metastasis models). However, systemic efficacy is limited by rapid clearance. All animal procedures require IACUC approval.

No detailed pharmacokinetic data are available for GRGDSP TFA. As a small linear hexapeptide (MW ~700 g/mol), GRGDSP is highly water-soluble and is rapidly cleared from the circulation when administered intravenously, with a plasma half-life of a few minutes (t1/2 < 5 min) due to glomerular filtration and proteolytic degradation by plasma peptidases. The peptide is not orally bioavailable. The TFA salt does not alter the PK properties. For in vivo applications, GRGDSP is often conjugated to a carrier (e.g., nanoparticles, PEG, or polymers) to prolong circulation time, enhance tumor accumulation, and improve efficacy. The conjugated forms have half-lives ranging from 1-8 hours depending on the carrier size and PEGylation. The free peptide is not used for therapeutic purposes, and detailed PK parameters are not published. GRGDSP is for research use only.

No specific toxicity data are available for GRGDSP TFA. As a short synthetic peptide composed of naturally occurring L-amino acids, GRGDSP is generally considered to have low toxicity. In vitro, GRGDSP is not cytotoxic to cells at concentrations up to 1 mM (48-72 hours), as assessed by MTT or LDH assays. In vivo, free GRGDSP administered intravenously at 50 mg/kg in mice is generally well-tolerated, with no acute mortality or significant weight loss reported in the literature. At very high doses (>100 mg/kg), transient hypotension and prolonged bleeding time may occur due to inhibition of platelet alphaIIbbeta3 integrins; however, these are pharmacological effects, not toxicities. The TFA salt is present in low, stoichiometric amounts and is considered non-toxic. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been performed. The peptide is for research use only and is not intended for human or therapeutic use. Standard laboratory safety precautions (gloves, lab coat) should be used. The GRGDSP peptide is not an approved drug.

[1]. Transarterial administration of integrin inhibitor loaded nanoparticles combined with transarterial chemoembolization for treating hepatocellular carcinoma in a rat model. World J Gastroenterol. 2016 Jun 7;22(21):5042-9.

[2]. Regulation of endothelial cell function by GRGDSP peptide grafted on interpenetrating polymers. J Biomed Mater Res A. 2007 Nov;83(2):423-33.

The RGD (Arg-Gly-Asp) peptide is one of the most widely used motifs in cell biology research. It was originally identified as the cell adhesion site in fibronectin by Pierschbacher and Ruoslahti in 1984. GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) is a synthetic hexapeptide that is widely used as a soluble inhibitor of integrin function. It is often used as a standard RGD peptide in cell adhesion, migration, and integrin signaling studies. The peptide is also used as a surface coating to promote cell adhesion when immobilized. The control peptide GRGESP (Gly-Arg-Gly-Glu-Ser-Pro) or GRADSP (Gly-Arg-Ala-Asp-Ser-Pro) is used as a negative control. The TFA salt is used to improve solubility and stability. GRGDSP is not a drug and has not received regulatory approval for any medical indication. It is for research use only. The peptide is typically supplied as a lyophilized powder, stored at -20degC, and reconstituted in water or PBS. Avoid repeated freeze-thaw cycles. GRGDSP is widely used in studies of cancer metastasis, angiogenesis, thrombosis, bone resorption, and wound healing.

C24H38F3N9O12

701.61

GRGDSP;91037-75-1

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, avoid exposure to moisture.

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

H2O :~125 mg/mL (~178.16 mM)

Solubility in Formulation 1: 50 mg/mL (71.26 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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