Angiopep-2 Modification

Creative Peptides provides Angiopep-2 modification services to enhance brain-targeted drug delivery by utilizing blood-brain barrier (BBB) penetration strategies. Our expertise includes Angiopep-2 conjugation with small molecules, biologics, nanocarriers, nucleic acids, and imaging agents to facilitate receptor-mediated transcytosis via the LRP-1 pathway. Through precise chemical synthesis and bioconjugation techniques, we ensure efficient BBB transport, improved drug stability, and enhanced therapeutic efficacy, offering tailored solutions for neurological drug development.

What is Angiopep-2?

Angiopep-2 is an artificially engineered peptide originally discovered in a systematic study of 96 analogues by high-throughput screening techniques. Using a blood-brain barrier model constructed from bovine cell matrix, the researchers screened out Angiopep-2 and verified its mechanism of cross-cell transport mediated by low-density lipoprotein receptor-associated protein 1 (LRP-1). Experiments showed that its trafficking process could be inhibited by low temperature, RAP protein and α2-macroglobulin, suggesting that LRP-1 was the main target. Further in vitro and in situ cerebral perfusion experiments confirmed that the trans-blood-brain barrier efficiency of Angiopep-2 was significantly higher than that of other ligands such as transferrin, and was not affected by P-glycoprotein inhibitors, excluding the interference of efflux pumps. This property makes it the first peptide carrier to be shown to efficiently cross the BBB and accumulate in the brain parenchyma.

The structure of Angiopep-2 is derived from the human Kunitz domain, and its 19-amino acid sequence (TFFYGGSRGKRNNFKTEEY) enables it to specifically bind to the LRP-1 receptor. LRP-1 is highly expressed in BBB endothelial cells and glioma cells, conferring dual targeting capabilities on Angiopep-2: targeting both BBB and brain tumors. Based on this, Angiopep-2 is widely used to construct drug conjugates (such as paclitaxel-bound ANG1005) and modify nanocarriers to enhance the efficiency of drug delivery in the brain. The study also found that its transport efficiency was affected by the tumor microenvironment, and the expression of LRP-1 was up-regulated under hypoxic and acidic conditions, which further improved the targeting in the treatment of glioblastoma. These properties make Angiopep-2 a promising delivery tool for the treatment of brain diseases.

Fig.1 Structure of angiopep-2-paclitaxel conjugate (ANG1005, GRN1005)^3,4.

What is Angiopep-2 Modification?

Angiopep-2 modification is a strategy to chemically or biotechnologically conjugate Angiopep-2 peptides to drugs, nanocarriers, or contrast agents, with the aim of enhancing the delivery efficiency of therapeutic or diagnostic molecules to brain tissue by taking advantage of its ability to target the BBB. Angiopep-2 specifically binds to low-density LRP-1 on the surface of BBB endothelial cells through the SRGKRN domain in its sequence, mimicking native ligand-triggered receptor-mediated endocytosis (RMT). Upon binding, LRP-1 initiates endocytosis, encapsulating Angiopep-2 and its conjugates into intracellular vesicles and transporting them across endothelial cells to the brain parenchyma.

Mechanism of Angiopep-2 Modification

The core mechanism of Angiopep-2 modification relies on its ability to specifically target the BBB and enable cross-barrier delivery of drugs or vehicles through receptor-mediated endocytosis (RMT).

Targets Specific Binding of the LRP1 Receptor

Angiopep-2 is derived from the optimized design of the Kunitz-type serine protease inhibitor domain, which specifically binds to the LRP1 receptor that is highly expressed on the surface of BBB endothelial cells and glioma cells. LRP1 is an endocytic receptor that is widely distributed in BBB endothelial cells and glioma cells, mediating endocytosis transport of ligands. Compared with other targeted molecules such as transferrin or lactoferrin, Angiopep-2 has a higher affinity for LRP1 and a brain penetration efficiency that is tens of times higher.

Receptor-Mediated Endocytosis (RMT) Mechanisms

Angiopep-2-modified nanocarriers (such as PLGA NPs, liposomes, quantum dots, etc.) bind to the LRP1 receptor and trigger the invagination of the cell membrane to form internalized vesicles, which enter endothelial cells in a receptor-mediated endocytosis (RMT) manner. This process avoids the tight junction limitation of BBBs and significantly enhances the transmembrane efficiency of nanoparticles. For example, the ability of Angiopep-2-modified PLGA NPs to penetrate BBB in the in vitro model was significantly better than that of the unmodified group, and the uptake of glioma cells (U87MG) was nearly 1-fold increased after Ag2S quantum dots were modified with Angiopep-2.

Dual Targeting with Intracellular Trafficking

Angiopep-2 not only targets BBB, but also acts directly on brain tumor cells. LRP1 is overexpressed in glioma cells, which enables the modified nanoparticles to be further internalized by tumor cells after penetrating the BBB, achieving "dual targeting". For example, Thera-ANG-cHANPs targeted both BBB and tumor cells in glioblastoma models, significantly increasing intracellular accumulation of the chemotherapy drug irinotecan.

Synergistic Grooming and Design Optimization

Angiopep-2 is often co-modified with other targeted molecules (e.g., TAT peptide, transferrin) to enhance penetration efficiency through synergistic effects. For example, Angiopep-2 co-modified nanovesicles with TAT peptides combine LRP1 targeting and cell penetration, overcoming the saturation effect of a single receptor. In addition, nanocarrier design parameters (e.g., particle size, ligation chemistry, ligand density) affect the targeting effect. The optimized Angiopep-2 modified dendritic macromolecule (4 ligands) had the highest accumulation in the brain.

Advanced Platform for Angiopep-2 Modification

Peptide Synthesis and Modification Technology

Creative Peptides utilizes advanced solid-phase peptide synthesis (SPPS) technology to efficiently synthesize and modify Angiopep-2. Our automated systems enable precise control over peptide sequences, lengths, and modifications, ensuring that each Angiopep-2 peptide meets the specific requirements for optimal blood-brain barrier penetration. We offer various post-synthetic modifications, including PEGylation, phosphorylation, and glycosylation, which improve peptide stability, solubility, and bioavailability, enhancing its therapeutic potential for targeted drug delivery to the brain.

Cutting-Edge Analytical and Purification Tools

To ensure the highest quality of Angiopep-2 peptides, Creative Peptides employs state-of-the-art analytical and purification instruments, including High-Performance Liquid Chromatography (HPLC), Mass Spectrometry (MS), and Nuclear Magnetic Resonance (NMR). HPLC is used for peptide purification, ensuring that only high-purity Angiopep-2 peptides are delivered. MS enables detailed molecular weight determination and structural verification, while NMR provides in-depth insights into peptide conformation, allowing for the precise assessment of modifications and ensuring the functional integrity of the peptide for BBB targeting.

Comprehensive Characterization and Quality Control

Creative Peptides integrates comprehensive quality control procedures into the modification process of Angiopep-2 to ensure that each peptide meets rigorous standards for research and therapeutic use. Our characterization platforms are equipped to assess the molecular integrity, modification efficiency, and functional activity of Angiopep-2. Using techniques such as MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) and circular dichroism (CD) spectroscopy, we confirm the structural and functional properties of Angiopep-2, ensuring it is optimized for effective BBB penetration and drug delivery applications.

Strategy of Angiopep-2 Modification

Angiopep-2 modification technology employs various chemical or biological methods to conjugate the Angiopep-2 peptide with drugs, carriers, or imaging agents, enabling efficient blood-brain barrier targeting and delivery.

Drug-Peptide Conjugation

Direct chemical conjugation is a classic strategy that covalently links Angiopep-2 to drug molecules. This strategy is categorized into:

Cleavable Linkers: Enzyme-sensitive peptides (e.g., cathepsin substrate sequence GGFLG) or acid-sensitive hydrazone bonds (which break in the lysosomal low-pH environment) ensure precise drug release within target cells.

Non-cleavable Linkers: Stable amide or thioether bonds rely on lysosomal degradation to release the drug, suitable for sustained-release therapies.

For example, Angiopep-2 conjugated with paclitaxel via an ester bond forms ANG1005, achieving cerebrospinal fluid concentrations over 10 times higher than free paclitaxel. Phase III trials show ANG1005 significantly prolongs progression-free survival (PFS) in glioblastoma and brain-metastatic breast cancer patients without increasing systemic toxicity, validating the advantages of direct conjugation in enhancing drug delivery and efficacy in the brain.

Nanocarrier Functionalization

Angiopep-2 surface modification on nanocarriers enhances BBB penetration and drug stability.

Liposomes: Angiopep-2 is conjugated via maleimide-thiol reactions to the liposome surface (e.g., for doxorubicin-loaded liposomes), increasing brain delivery efficiency by fivefold.

Polymeric Nanoparticles: Poly (lactic-co-glycolic acid) (PLGA) or chitosan particles are conjugated via amine-carboxyl condensation to deliver genes or proteins (e.g., neurotrophic factor BDNF).

Exosomes: Genetic engineering embeds Angiopep-2 in exosomal membrane proteins to enhance brain penetration (e.g., for Aβ antibody delivery in Alzheimer's disease).

Nanocarriers protect drugs from enzymatic degradation and immune clearance while prolonging drug effects. For instance, Angiopep-2-modified PLGA nanoparticles increase doxorubicin brain concentration 15-fold while reducing cardiotoxicity.

Gene/Nucleic Acid Complexation

Angiopep-2-modified gene vectors provide new strategies for brain gene therapy.

Electrostatic Adsorption: Cationic carriers (e.g., polyethyleneimine, PEI) electrostatically bind Angiopep-2 and nucleic acids (e.g., siRNA) to form stable complexes.

Covalent Conjugation: Angiopep-2 is directly linked to gene carriers (e.g., lipid nanoparticles) to enhance targeting and stability.

For example, Angiopep-2-modified siRNA complexes selectively silence the oncogene EGFR in glioblastoma, reducing tumor volume by 70% in animal models.

Imaging Agent Modification

Angiopep-2-modified imaging agents enhance brain lesion imaging accuracy.

Gadolinium-Based Nanoparticles: Angiopep-2-modified superparamagnetic iron oxide nanoparticles (SPIONs) improve T1-weighted MRI contrast of brain tumors, increasing sensitivity threefold.

Near-Infrared Fluorescence Probes: Angiopep-2 conjugated with Cy5.5 dye enables real-time monitoring of brain metastasis vascular leakage and drug distribution, guiding precise treatment.

These modified imaging agents improve diagnostic accuracy and can be combined with therapeutic carriers for theranostic applications, such as intraoperative fluorescence navigation coupled with post-surgical targeted chemotherapy.

Multimodal Conjugation

Integrating Angiopep-2 with other functional modules enables synergistic delivery and smart drug release.

Dual-Target Modification: Co-modification of Angiopep-2 and transferrin receptor (TfR) ligands on liposomes enhances BBB penetration via dual LRP-1 and TfR pathways, increasing transcytosis efficiency by 40%.

Stimuli-Responsive Carriers: Angiopep-2-modified pH-sensitive nanoparticles rapidly release drugs in the acidic tumor microenvironment or ROS-sensitive linkers trigger drug release at inflammation sites.

Multimodal designs overcome single-target limitations. For example, co-modification of Angiopep-2 with cell-penetrating peptides (CPPs) improves drug diffusion in brain parenchyma, offering a promising approach for diffuse glioma treatment.  

Advantages of Our Angiopep-2 Modification Service

Application of Angiopep-2 Modification

Customized Service Process

FAQs

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References

1. Habib, Saffiya, and Moganavelli Singh. Angiopep-2-modified nanoparticles for brain-directed delivery of therapeutics: a review. Polymers 14.4 (2022): 712.
2. Li, Han, et al., Angiopep-2 modified exosomes load rifampicin with potential for treating central nervous system tuberculosis. International Journal of Nanomedicine (2023): 489-503.
3. Image retrieved from Figure 3 "Chemical structure of angiopep-2-paxlitaxel conjugate (ANG1005, GRN1005)," He, Rongjun, et al., 2019, used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The title was changed to "Structure of angiopep-2-paclitaxel conjugate (ANG1005, GRN1005)".
4. He, Rongjun, et al., Peptide conjugates with small molecules designed to enhance efficacy and safety. Molecules 24.10 (2019): 1855.