How Glycopeptides Enhance Targeted Drug Delivery: A Practical Guide for Researchers?

What Are Glycopeptides and Why Use Them in Drug Delivery?

(1) Glycopeptides as Dual-Function Molecules (Ligand + Carrier)

Glycopeptides (GPs) are a bioconjugate that have both the characteristics of peptides as a desired functional group or ligand and glycans as a desired targeting agent. GPs have the dual properties to function as both a carrier and ligand. The carrier glycan may allow for a higher stability and solubility of the bioconjugate and the ligand peptide may allow for recognition or binding of a certain protein or cell. Glycoproteins can serve as ligands for applications but also target specific tissue or cell constructs therapeutically. Previous reports show that GPs capable of binding to the asialoglycoprotein receptor (ASGPR) can be efficiently internalized by hepatocytes in addition to cell lines which overexpress this receptor including hepatocellular carcinoma and cholangiocarcinoma cells. Attempts have also been made to covalently attach a therapeutic agent to a GP in order to increase the delivery of the agent to liver cells, and in turn increase the efficacy and decrease the off target toxicity. The half-life of a conjugate can also be increased by the glycosylation of peptides by protecting them from proteolytic enzymes.

(2) Glycan-Mediated Targeting to Receptors (e.g. ASGPR, MGL, CD22)

The glycan molecules operate as ligands for multiple receptors including ASGPR, mannose receptor (MGL), and CD22 which are found on different immune and cancer cells. GPs engineered with specific glycan receptors enhance targeted cell drug delivery while improving therapeutic effectiveness and minimizing systemic side effects. For example, studies have demonstrated that GPs that target the ASGPR can efficiently deliver drugs to hepatocytes and certain cancer cells that overexpress this receptor. GPs have also been designed to target the MGL, which is expressed on dendritic cells and macrophages, which play important roles in the immune system. Targeting these cells using GPs can significantly enhance the bioavailability and therapeutic index of the drugs that are conjugated to the glycopolymers.

Fig. 1 Strategies based on targeting cancer-associated glycans for selective and specific drug delivery systems. ^1,2

Advantages of Glycopeptides in Targeted Therapeutics

(1) Increased Solubility and Circulation Time

Improved solubility and an extended circulation time are among the most critical pharmacokinetic advantages of GPs. By increasing the overall hydrophilicity of the parent molecule, the addition of the glycan moiety to the peptide backbone leads to enhanced aqueous solubility. Peptides are characterized by poor aqueous solubility and a high clearance rate in the bloodstream, which severely limits their use. The addition of glycosylation moieties have the potential to significantly enhance stability and bioavailability of peptide therapeutics. GPs have an extended half-life in the blood. In a study comparing serum half-life of glycosylated and non-glycosylated proteins, it was found that glycosylated proteins could have a fourfold longer time in serum circulation. The extended systemic circulation of the drug allows for the reduction in dosing frequency and the maintained serum levels may increase patient compliance and lower the incidence of adverse effects. GPs are more stable against proteolytic degradation and are less easily cleared by the kidneys than their non-glycosylated analogues. GPs have a relatively lipophilic backbone that will interact with the membrane surface in an α-helical conformation while the carbohydrate moiety increases water solubility and interacts with the aqueous compartment in an ensemble of random coil conformations. This allows the peptide to interact with the membrane surface and aqueous compartment simultaneously through a "hopping" motion, which increases bioavailability by reducing an inefficient three-dimensional search for its target receptor to a simpler two-dimensional search, ultimately decreasing the amount of space the peptide must survey.

(2) Site-Specific Targeting via Glycan Recognition

An additional advantage of using GPs as conjugation platforms for targeted therapeutics is the ability to target specifically through glycan recognition. Glycans serve as ligands for a variety of receptors. This cell-selective targeting ability has been shown by GPs conjugated to drugs that target receptors overexpressed on cancer cells. For example, GPs that target ASGPR have been used for efficient drug delivery to hepatocytes and certain cancer cells that overexpress ASGPR, while GPs designed to target MGL can deliver drugs to dendritic cells and macrophages. This targeted drug delivery has been shown to improve the therapeutic index of the conjugated drug by reducing off-target side effects.

Real-World Use Cases: Glycopeptides in Nanocarriers and Conjugates

(1) Liposome Functionalization with Glycopeptides

Liposomes can be enhanced with GPs to improve their targeting abilities and therapeutic effects. GPs have the capability of being altered to enable targeting specific tissues or cells within the body. The system enables specific delivery of pharmaceutical drugs and therapeutic substances to their required destination. By attaching GPs to liposomes researchers can specifically target ASGPR which appears in high amounts on hepatocytes as well as certain cancer cells. GP-functionalized liposomes serve as vehicles to direct drugs and therapeutic agents towards their target cells. One potential application of glycopeptide-functionalized liposomes is the use of glycopeptides to target ASGPR on hepatocytes. ASGPR is a receptor that is overexpressed on the surface of hepatocytes. GPs can be conjugated to liposomes to target ASGPR, which can allow for the selective delivery of drugs or other therapeutic agents to hepatocytes. A research study demonstrated the synthesis of glycopeptide-functionalized liposomes which were utilized to target hepatocytes with the chemotherapeutic drug doxorubicin (DOX). The glycopeptide-functionalized liposomes showed improved cellular uptake and antitumor efficacy in cancer models compared to non-functionalized liposomes. The GPs on the liposomes surface enhanced hepatocyte targeting while increasing liposome stability and blood circulation time to decrease off-target effects and systemic toxicityx.

(2) Glycopeptide–Drug Conjugates (GPDCs) in Preclinical Research

Researchers have utilized GPDCs as a preclinical research method to enhance molecule targeting accuracy and boost their therapeutic effectiveness. GPDCs have been evaluated for application in cancer therapy. The conjugation of chemotherapeutic drugs to glycopeptides results in a drug delivery system that targets cancer cells and can increase the therapeutic index of a chemotherapeutic drug by increasing its therapeutic efficacy and reducing toxicity to non-targeted cells. In one study, a GPDC was designed to deliver doxorubicin to cancer cells overexpressing a specific receptor. The GPDC exhibited higher cellular uptake and antitumor efficacy in a preclinical cancer model compared to free doxorubicin, with reduced systemic toxicity. The glycopeptide portion of the conjugate functioned as a ligand to specifically target cancer cells, thereby directing the drug to the tumor site. The design of GPDCs aims to optimize both the selectivity and effectiveness of antimicrobial treatments. By linking an antibiotic to a glycopeptide which binds bacterial receptors a GPDC emerges that targets bacterial cells exclusively to boost antibiotic effectiveness and minimize effects on non-target cells. In one study, a GPDC was developed to combat drug-resistant bacterial infections. The GPDC retained antibacterial activity in the presence of high salt concentrations, where most antimicrobial peptides are inactive. This targeted delivery approach can also reduce the development of antibiotic resistance.

How We Support Targeted Delivery Projects with Glycopeptides?

(1) Custom Synthesis of Ligand Glycopeptides

Our custom synthesis services for ligand GPs are designed to meet the specific needs of targeted delivery projects. We specialize in creating highly defined and homogeneous GPs that can be tailored to interact with specific receptors or cellular targets. Our synthesis strategies include chemoselective ligation of oligosaccharides to peptides, traditional peptide synthesis with unprotected glycosylated amino acids, and enzymatic glycan extension. Our service capabilities cover the full spectrum of glycopeptide synthesis including: N-linked and O-linked GPs, highly complex glycoforms with various glycosylation sites. Our expert team can also design and synthesize GPs with specific glycan structures, such as targeting the ASGPR or other receptors for targeting cancer cells or immune cells. GPs from our custom synthesis services can be made with state of the art instrumentation and first class synthesis technology to provide high quality, high purity final products. We can incorporate other modifications such as fluorescent labels or stable isotopes, allowing for easy tracking and analysis of the GPs in biological systems.

(2) Analytical and Characterization Services for Conjugates

Our company provides characterization and analytical services that support glycopeptide conjugate development and optimization together with custom synthesis. The process requires structural analysis of synthesized GPs and conjugates to verify their chemical structure, purity and molecular integrity. It can be carried out using multiple approaches including HPLC, MS, etc. Glycopeptide conjugates can also have glycan profiling to identify the glycan structures present in the conjugate and their relative abundance. This can be used to ensure the desired glycosylation pattern has been achieved and can also provide insight into potential efficacy of the conjugate. Stability testing of glycopeptide conjugates under various conditions can also be performed to assess their long-term stability and integrity. Custom analytical methods can be constructed based on specific project requirements which include quantitative assays for glycopeptide conjugates and assessments of binding affinity and specificity to target receptors.

Peptide Conjugation Services at Creative Peptides

References

1. Image retrieved from Figure 1 " Strategies based on targeting cancer-associated glycans for selective and specific drug delivery systems," Diniz F.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. Diniz F.; et al. " Glycans as targets for drug delivery in cancer." Cancers, 2022, 14(4): 911.
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4. Li Q, Guo Z. Recent advances in toll like receptor-targeting glycoconjugate vaccines[J]. Molecules, 2018, 23(7): 1583. https://doi.org/10.3390/molecules23071583.
5. Guo Y, Jia W, Yang J, et al. Cancer glycomics offers potential biomarkers and therapeutic targets in the framework of 3P medicine[J]. Frontiers in endocrinology, 2022, 13: 970489. https://doi.org/10.3389/fendo.2022.970489.