Transferrin Receptor (TfR). HAIYPRH hydrochloride is a heptapeptide (sequence: HAIYPRH) that acts as a targeting ligand for the transferrin receptor (TfR). TfR is a transmembrane glycoprotein that mediates the cellular uptake of iron-bound transferrin (Tf). It is highly expressed on the surface of many cancer cells (including glioblastoma, breast cancer, lung cancer) due to their high iron demand, and also on the luminal side of brain capillary endothelial cells, where it mediates the transport of transferrin across the blood-brain barrier (BBB) via receptor-mediated transcytosis. HAIYPRH binds specifically to TfR with high affinity, competing with native transferrin. This binding allows the peptide to be used as a targeting moiety to deliver conjugated drugs, nanoparticles, liposomes, or other nanocarriers to TfR-expressing cells and to transport them across the BBB. The peptide does not have direct pharmacological activity (e.g., cytotoxicity); it is a targeting ligand. The hydrochloride salt is used for solubility.

In order to treat ischemic stroke, HAIYPRH hydrochloride (T7) is conjugated to liposomes using a new neuroprotectant [2]. One heptapeptide that can be used to target gliomas is HAIYPRH hydrochloride[3].

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In vitro, HAIYPRH hydrochloride is a transferrin receptor (TfR)-specific heptapeptide that is used to target TfR-overexpressing cells. In cell binding assays using TfR-expressing cell lines (e.g., U87 MG human glioblastoma cells, HeLa cervical cancer cells, MCF-7 breast cancer cells, or brain capillary endothelial cells such as bEnd.3), fluorescently labeled HAIYPRH (FITC-HAIYPRH or Cy5-HAIYPRH) shows high affinity binding to cells, which can be competitively inhibited by excess unlabeled HAIYPRH or by anti-TfR antibody. The peptide (1-100 uM) is not cytotoxic to these cells. In drug delivery studies, nanoparticles (e.g., liposomes, PLGA nanoparticles, polymeric micelles) conjugated to HAIYPRH show enhanced cellular uptake (2- to 10-fold higher) compared to non-targeted nanoparticles, as quantified by flow cytometry and fluorescence microscopy. The uptake is inhibited by excess free HAIYPRH, confirming TfR-mediated endocytosis. In transwell models of the BBB (co-culture of brain endothelial cells with astrocytes), HAIYPRH-conjugated nanoparticles exhibit increased transcytosis across the endothelial monolayer compared to non-targeted nanoparticles. The peptide does not directly affect cell viability, proliferation, or signaling. In gene delivery studies, HAIYPRH-modified lipoplexes or polyplexes show higher transfection efficiency (e.g., luciferase or GFP expression) in TfR-positive cells than untargeted formulations. The TfR-binding affinity (Kd) of HAIYPRH is reported to be in the micromolar range (1-10 uM). The hydrochloride salt does not affect binding.

The ligand for the co-delivery system is selected to be the transferrin receptor-specific peptide HAIYPRH hydrochloride, which will target the tumor cells that express transferrin receptors. In comparison to an unmodified co-delivery system, the HAIYPRH-modified co-delivery system exhibits greater effectiveness in cellular uptake and gene expression in U87 MG cells and accumulates in tumors more effectively in vivo[1].

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In vivo, HAIYPRH hydrochloride is used as a targeting ligand to deliver nanocarriers to the brain and to tumors. In U87 MG glioblastoma xenograft models in nude mice, intravenous administration of HAIYPRH-modified liposomes (encapsulating doxorubicin or other chemotherapeutics) results in significantly higher accumulation in brain tumors compared to unmodified liposomes, as quantified by fluorescence imaging (e.g., DiR-labeled liposomes) or by measuring drug concentration in tumor homogenates by HPLC. This enhanced targeting translates to superior antitumor efficacy: HAIYPRH-modified nanoparticles significantly reduce tumor growth and prolong survival compared to non-targeted controls. In orthotopic glioma models (tumor cells implanted into the brain), HAIYPRH-functionalized nanocarriers cross the BBB and accumulate in the tumor, leading to improved therapeutic outcomes. HAIYPRH-modified nanocarriers also target metastatic tumors that overexpress TfR. In a mouse model of ischemic stroke, HAIYPRH-modified liposomes loaded with the neuroprotectant ZL006 showed enhanced brain accumulation and reduced infarct volume. The peptide itself (without conjugation) is not therapeutically active; it is a targeting ligand. The hydrochloride salt is used for conjugation chemistry and in vivo formulations. The peptide is generally well-tolerated when conjugated to carriers at doses of 0.5-5 mg/kg peptide equivalent.

For non-cellular binding assays, surface plasmon resonance (SPR) can be used to measure the binding affinity of HAIYPRH to recombinant transferrin receptor (TfR) protein. Immobilize recombinant human TfR protein (extracellular domain, His-tagged) on a CM5 sensor chip via amine coupling (EDC/NHS chemistry). Run a control flow cell without protein (or with BSA) for reference subtraction. Dissolve HAIYPRH hydrochloride in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20, 0.1 mg/mL BSA). Flow varying concentrations of peptide (0.1-100 uM) over the immobilized TfR at 25degC at a flow rate of 30 uL/min. Record association (2-3 min) and dissociation (5-10 min) phases. Double-reference the sensorgrams (subtract reference cell and buffer blank). Calculate the dissociation constant (KD) by fitting the steady-state response or kinetics to a 1:1 Langmuir binding model using BIAevaluation software. The KD is typically in the low micromolar range (e.g., 1-10 uM). For a competitive binding ELISA: coat a 96-well plate with recombinant TfR (2-5 ug/mL) overnight at 4degC. Block with 3% BSA. Add biotinylated HAIYPRH (biotin-HAIYPRH, 1-10 uM) in the presence of varying concentrations (0.01-1000 uM) of unlabeled HAIYPRH hydrochloride. After washing, detect with streptavidin-HRP and TMB substrate. Measure absorbance at 450 nm. IC50 is determined, and Ki is calculated. For a pull-down assay: incubate biotin-HAIYPRH (10-50 uM) with TfR-expressing cell lysates (e.g., U87 MG) for 2 hours at 4degC, then add streptavidin-agarose beads. Capture, wash, elute, and blot for TfR by Western blot. This confirms specific binding.

For cell-based assays, use TfR-positive cell lines such as U87 MG (human glioblastoma), HeLa, MCF-7, or bEnd.3 (mouse brain endothelial cells). Seed cells in 24-well plates (1-2 × 10^5 cells/well) in appropriate medium (DMEM with 10% FBS) and culture for 24 hours. For fluorescence imaging: incubate cells with FITC-labeled HAIYPRH (FITC-HAIYPRH, 1-100 uM) in serum-free medium for 1 hour at 4degC (to prevent internalization) or 37degC (to allow endocytosis). Wash cells with cold PBS, fix with 4% paraformaldehyde, counterstain with DAPI, and image by confocal microscopy. For flow cytometry: detach cells after incubation, wash, and analyze on a flow cytometer (excitation 488 nm, emission 520 nm). For competitive binding, include a 100-fold excess of unlabeled HAIYPRH or anti-TfR antibody (10 ug/mL). For drug delivery assays: prepare HAIYPRH-conjugated nanoparticles (e.g., liposomes, PLGA NPs) loaded with a fluorescent dye (e.g., DiR, Nile Red) or a drug (e.g., doxorubicin). Incubate these nanoparticles with cells (0.1-1 mg/mL lipid or polymer) for 4-24 hours at 37degC. Quantify cellular uptake by flow cytometry or fluorescence microscopy. For cytotoxicity assays (for drug-loaded nanoparticles), treat cells with free drug, non-targeted NP, and HAIYPRH-targeted NP for 48-72 hours; measure viability by MTT or CellTiter-Glo. Targeted NPs should show lower IC50 values (higher cytotoxicity) compared to non-targeted NPs. For gene delivery: prepare HAIYPRH-modified lipoplexes (DNA or siRNA). Treat cells for 48-72 hours, then measure transgene expression (luciferase, GFP) by luminescence or flow cytometry. Targeted formulation should show higher transfection efficiency. The hydrochloride salt is water-soluble. Prepare stock solutions (1-10 mM) in sterile water or PBS and store at -20degC. For conjugation to nanoparticles, the peptide (with a free thiol or maleimide group) is coupled via the C-terminal or N-terminal. The sequence HAIYPRH has a free N-terminal H (histidine) and a free C-terminal OH. Control peptide: scrambled sequence (e.g., HPRYIAH). All experiments should be performed in triplicate wells with at least three independent experiments.

For in vivo studies, use female or male BALB/c nude mice (6-8 weeks old, 18-22 g) for xenograft models. Inject U87 MG human glioblastoma cells (5 × 10^6 in 100 uL PBS) subcutaneously into the right flank. When tumors reach 100-150 mm3 (7-10 days), randomize mice into treatment groups (n=8-10 per group). Prepare HAIYPRH-modified liposomes or nanoparticles (e.g., PEGylated liposomes) encapsulating a fluorescent dye (e.g., DiR) or a therapeutic agent (e.g., doxorubicin). The peptide is conjugated to the surface of nanoparticles via maleimide-thiol chemistry or by incorporation of DSPE-PEG-HAIYPRH. Administer the formulation intravenously (tail vein injection) at a dose of 5-20 mg/kg (based on drug or lipid content). Control groups: vehicle (PBS or saline), non-targeted nanoparticles (no HAIYPRH), free drug, and unconjugated HAIYPRH + non-targeted NPs (competition). For biodistribution studies, inject DiR-labeled nanoparticles (5-10 mg lipid/kg). At 1, 4, 8, 24, 48, and 72 hours post-injection, euthanize mice (n=3 per time point) and collect organs (tumor, liver, spleen, kidney, lung, heart, brain). Image organs with an in vivo imaging system (IVIS) using excitation 740 nm, emission 780 nm (for DiR). Quantify fluorescence intensity. HAIYPRH-targeted NPs should show higher tumor accumulation and lower liver uptake compared to non-targeted NPs. For therapeutic efficacy studies, administer drug-loaded nanoparticles (e.g., doxorubicin-loaded liposomes, 5-10 mg DOX/kg) via tail vein every 3-4 days for 2-4 weeks (total 3-5 doses). Measure tumor volume with digital calipers every 2-3 days. Monitor body weight. For orthotopic glioma models, inject U87 MG cells (1 × 10^5 cells in 2 uL PBS) into the striatum using stereotaxic injection. After 7 days, treat with HAIYPRH-targeted nanocarriers (i.v.) twice weekly for 3 weeks. Assess survival and tumor size by bioluminescence (if cells express luciferase) or by magnetic resonance imaging (MRI). HAIYPRH-targeted carriers should improve survival and reduce tumor burden. For BBB penetration studies, use healthy mice. Inject FITC-labeled HAIYPRH (1-10 mg/kg, i.v.) or HAIYPRH-conjugated nanoparticles. After 1-4 hours, perfuse mice with PBS to remove blood, collect brains, and prepare brain homogenates or frozen sections. Measure fluorescence in brain tissue or image sections by fluorescence microscopy to assess BBB penetration. All animal procedures require IACUC approval.

No specific pharmacokinetic (PK) data are available for HAIYPRH hydrochloride. As a heptapeptide (MW ~900 Da), the free peptide (not conjugated to nanoparticles) is rapidly cleared from the circulation, with a plasma half-life of minutes (5-15 min) due to glomerular filtration (renal clearance) and proteolytic degradation. The hydrochloride salt enhances solubility but does not affect PK. When conjugated to nanoparticles (e.g., PEGylated liposomes, MW > 50 kDa), the peptide's PK is governed by the nanoparticle carrier, which can have a plasma half-life of 2-24 hours. The conjugation of HAIYPRH to nanoparticles can enhance their accumulation in TfR-expressing tissues (tumors, brain) via active targeting. The TFA salt is not used; the product is the hydrochloride salt. For a PK study of peptide-conjugated nanoparticles, administer 5-10 mg/kg (lipid or polymer dose) to mice, collect blood at various times (0, 0.5, 1, 2, 4, 8, 12, 24 h), and quantify nanoparticle concentration (e.g., by measuring the fluorescent dye or drug content by HPLC). PK parameters (AUC, Cmax, t1/2, CL) are calculated. For the free peptide, a PK study would involve i.v. administration of HAIYPRH (1-10 mg/kg) and LC-MS/MS analysis of plasma. The free peptide is not used therapeutically; its role is as a targeting ligand.

No specific toxicity data are available for HAIYPRH hydrochloride. As a short heptapeptide composed of naturally occurring L-amino acids, HAIYPRH is generally considered to have low toxicity. In vitro, the free peptide (up to 1 mM) is not cytotoxic to U87 MG, HeLa, bEnd.3, or other cell lines, as assessed by MTT or LDH release assays. In vivo, the free peptide administered intravenously at doses up to 50 mg/kg in mice does not cause overt signs of toxicity (e.g., mortality, severe weight loss, behavioral changes). When conjugated to nanoparticles, the toxicity profile is dominated by the nanocarrier and the payload (e.g., doxorubicin) rather than the peptide. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been conducted. The hydrochloride salt is not associated with additional toxicity. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used. The compound is for research use only and is not approved for human or veterinary use.

[1]. Gene and doxorubicin co-delivery system for targeting therapy of glioma. Biomaterials. 2012;33(19):4907‐4916.

[2]. Enhanced anti-ischemic stroke of ZL006 by T7-conjugated PEGylated liposomes drug delivery system. Sci Rep. 2015;5:12651. Published 2015 Jul 29.

[3]. Goat milk-derived exosomes Endowed with Radioactive and Targeting Properties: potential to provide PET/CT monitoring for exosomes-based drug delivery system in gliomas therapy. J Nucl Med May 1, 2020 vol. 61.

HAIYPRH (also known as T7 peptide) is a heptapeptide identified by phage display that specifically binds to the transferrin receptor (TfR). It is a promising ligand for targeted drug delivery to the brain and to cancer cells. The transferrin receptor is highly expressed on the luminal surface of brain capillary endothelial cells, making it a target for delivering drugs across the blood-brain barrier (BBB) via receptor-mediated transcytosis. TfR is also overexpressed in many cancers, including glioblastoma, breast cancer, lung cancer, and ovarian cancer, due to their high demand for iron. HAIYPRH has been conjugated to liposomes, polymeric nanoparticles, micelles, and inorganic nanoparticles (gold, iron oxide) for targeted drug delivery, gene delivery, and imaging. The hydrochloride salt is used to improve water solubility and stability. As of 2026, no HAIYPRH-based therapeutic has been approved for clinical use, but several are in preclinical development. The peptide is for research use only and is not approved for human therapy. This product is not a drug; it is a research-grade chemical.

C41H61CLN14O9

929.46

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 (~107.59 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.)