Custom Glycopeptides for Targeted Nanoparticle Delivery Systems

Why Functionalize Nanoparticles with Glycopeptides?

(1) Improved Bioavailability and Cellular Uptake

Functionalization of nanoparticles (NPs) with glycopeptides can also affect their bioavailability and cellular uptake. The targeting of ligands to the surface of specific receptors allows for targeted delivery of NPs. Glycopeptides can overcome some of the limitations associated with NP absorption when administered orally. Glycopeptide-functionalized NPs can be engineered to target surface receptors that are overexpressed on hepatocytes and the surface of certain cancer cells. The attachment of glycopeptides to NPs allows these particles to bind with specific cell surface receptors thereby enhancing cellular uptake and boosting therapeutic delivery. In addition to their impact on NP uptake, glycopeptides can also modulate the stability and solubility of nanoparticles, which can impact their bioavailability. For instance, PEGylation of the surface of NPs can increase their stability and decrease their immunogenicity. Glycopeptides can also improve the interaction of NPs with the intestinal mucosa, which can enhance their uptake and absorption.

Fig. 1 Design of a liposomal vaccine consisting of auxiliary lipids, MUC1 lipoglycopeptides and MPLA adjuvant.^1,2

(2) Reduction of Off-Target Effects

Another application for glycopeptide-functionalized NPs is to decrease off-target effects. Glycopeptides are able to bind specific receptors on specific cells so they can be used to target the nanoparticles to these specific cells and avoid off-target effects on other cells and tissues. Lectins are glycoproteins that bind specifically to sugars resulting in agglutination and precipitation amongst cells and glycoconjugates, but do not break covalent linkages. Lectin can also be covalently conjugated to nanoparticles. Lectin conjugation to NPs through interaction with mucus or the intestinal epithelial cell surface may aid nanoparticles in crossing the intestinal mucosa. A poly-lactide-co-glycolide NPs (PLGA-NPs) that was engineered to have a glycopeptide (g7), a polymeric matrix structure (rigid) and decorated with an amphiphatic g7 with a helix-like conformation was able to cross the blood brain barrier in vivo and have the ability to interact with neuronal cells (mainly neurons) and was properly evaluated in vitro on cell cultures.

Strategies for Surface Conjugation

(1) Covalent vs Non-Covalent Attachment

Covalent bonds are strong and stable chemical bonds that occur when atoms share electrons. This method is generally used for applications that require stable and long-lasting conjugates. EDC-NHS crosslinking serves as a standard method to covalently attach biomolecules. EDC and NHS facilitate biomolecule surface attachment by forming stable amide bonds between carboxyl groups on the surface and amine groups on the biomolecule. The high stability of covalent bonds make this a suitable method for applications where the stability of the conjugate is essential, for example targeted drug delivery. Non-covalent bonds are generally far weaker and more transient than covalent bonds. The two primary types of non-covalent bonds are hydrogen bonds, van der Waals forces, and electrostatic interactions. Benzophenone photochemistry is a non-covalent attachment technique that immobilizes biomolecules on surfaces with UV light. This method offers precise control over the attachment process and can be used to pattern immobilized biomolecules. Non-covalent interactions are particularly useful for applications where the biomolecule needs to be released or when the surface needs to be functionalized with multiple biomolecules.

(2) Spacer Arm Optimization for Glycopeptide Exposure

Optimizing spacer arms can lead to the maximum possible exposure of glycopeptides on the nanoparticle surface. Spacer arms directly affect glycopeptide orientation and stability which then determines their binding affinity and targeting specificity toward receptors. The length and flexibility of the spacer arms are important design parameters that can be optimized to improve the exposure of glycopeptides. Polyethylene glycol chains maintain appropriate separation between the glycopeptides and nanoparticle surfaces to lessen steric hindrance while boosting the potential for receptor binding. Selecting the appropriate spacer arm determines the conjugate's hydrophilicity which affects solubility and decreases non-specific binding. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can be used to quantify the binding affinity and specificity of glycopeptides with different spacer arms, which can provide information about the optimal spacer arm design for maximizing receptor binding and minimizing off-target effects. For example, it has been reported that PEGylated spacers can significantly enhance the stability and bioavailability of glycopeptide-conjugated NPs.

Case Study: Glycopeptide-Coated Liposomes in Hepatic Targeting

(1) ASGPR Ligands and Their Effectiveness

Liver targeting glycopeptide-coated liposomes have been investigated through the ASGPR. ASGPR is a C-type lectin receptor, that is well characterized for its recognition and binding to galactose and GalNAc residues in glycoproteins and its subsequent endocytosis and degradation in hepatocytes. Glycopeptides targeted to the ASGPR have been determined to be highly successful. In studies, it was shown that liver-selective ASGPR ligands increased the hepatocyte-uptake of the targeted liposomes. Liposomes were functionalized with glycopeptides containing GalNAc residue resulted in a significantly higher hepatocyte uptake when compared to non-functionalized liposomes. The binding of ASGPR with sugar residues Gal/GalNAc was demonstrated by flow cytometry and confocal microscopy analysis, confirming that ASGPR has high affinity for this glycan of interest. Targeting with ligands of ASGPR for hepatic accumulation in the liver has been confirmed in vitro and in vivo. The ASGPR ligands functionalized on the surface of NPs and internalized in vitro with hepatocyte cell lines have exhibited results of receptor-mediated endocytosis. The hepatic selective nanoparticles are internalized through receptor-mediated endocytosis and the targeting functionality results in the delivery of payload in the lumen of hepatocytes. Nanocarriers that are chemically inert and have tunable size and shape for easy conjugation of antigens on their surface with disulfide bonds between particles and immunogens are possible. Anticancer therapy has been studied with iron oxide NPs coated with TACA conjugated with phospholipid, an example of glycopeptide. Multivalent glycopeptide-coated iron oxide NPs were shown to augment the interaction with B cells through multiple mechanisms of binding. The NPs also induced strong antibody responses that were able to recognize glycopeptide-expressing tumors and killed tumor cells through complement-mediated cytotoxicity.

In Vivo vs In Vitro Performance Data

The in vitro and in vivo targeting efficacy of glycopeptide-coated liposomes to the liver has been reported extensively. In vitro, glycopeptide-coated liposomes have been shown to have high targeting specificity and efficiency to hepatocytes. For instance, cell uptake assays have been used to demonstrate that glycopeptide-coated liposomes are rapidly internalized by hepatocytes and accumulate in the cells. In vivo, the targeting efficacy of glycopeptide-coated liposomes to the liver has also been evaluated in animal models. These studies have generally shown that glycopeptide-coated liposomes have enhanced hepatic targeting in vivo compared to non-functionalized liposomes. For example, studies in rats have shown that glycopeptide-coated liposomes accumulate predominantly in the liver with minimal distribution to other organs. This selective uptake is a result of high expression levels of the ASGPR on hepatocytes and the specific interaction between the glycopeptide ligands and the ASGPR. In addition, in vivo experiments have demonstrated that glycopeptide coated liposomes display prolonged circulation times compared to non-functionalized liposomes. This could be related to a shielding effect provided by the glycopeptide coating which can decrease clearance by the reticuloendothelial system. The combination of increased hepatic targeting and prolonged circulation times should result in an improved therapeutic efficacy of these liposomes as drug delivery vehicles to the liver.

How We Help: Custom Glycopeptides for Nanocarrier R&D?

(1) Solubility Tuning and Surface Charge Control

Solubility tuning and surface charge control are two additional design considerations that should be taken into account. Nanocarrier delivery effectiveness to target sites depends on solubility tuning. Surface charge control is necessary to control the interaction between the nanocarrier and the target cells or tissues. Surface modification can be used to tune the solubility of nanocarriers. Surface functionalization of nanoparticles with glycopeptides can introduce hydrophilic regions on the surface of the carrier, which can improve its solubility in aqueous environments. The carrier's solubility plays a vital role in determining the bioavailability of poorly soluble drugs. PEGylation serves as a standard surface modification method that greatly enhances nanoparticle solubility and stability. Custom glycopeptides can be designed with PEG-like spacers to provide similar solubility and stability effects. Surface charge can be controlled through the use of glycopeptides. Surface charge can impact the targeting efficiency of the nanocarrier and reduce non-specific binding. For example, a negative surface charge can help prevent aggregation and improve the colloidal stability of the nanoparticles. The design of custom glycopeptides enables precise control over nanocarrier surface charges which enhances target cell interaction while minimizing off-target effects.

(2) Pre-assembled Glycopeptide-Linker Systems

We offer pre-assembled glycopeptide-linker systems with optimized linkers that are pre-engineered to orient and expose the glycopeptide on the nanocarrier surface. The linkers are designed to minimize steric hindrance and maximize receptor binding. Advantages of using pre-assembled glycopeptide-linker systems include saving time and effort in linker design, allowing researchers to focus on the development of their targeted delivery system. We perform extensive testing of our pre-assembled systems to ensure they have high binding affinity and specificity to target receptors such as ASGPR. Our customization service also caters to those researchers that need unique glycopeptides and linkers for their desired target receptors. From an array of glycopeptides and linkers, researchers can select and design their own custom system for optimal functionality in their desired application such as hepatic targeting, cancer therapy, etc.

Glycoamino acids we can provide

References

1. Image retrieved from Figure 1 " Design of a liposomal vaccine consisting of auxiliary lipids, MUC1 lipoglycopeptides and MPLA adjuvant," Du J J.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. Du J J.; et al. " MUC1 specific immune responses enhanced by coadministration of liposomal DDA/MPLA and lipoglycopeptide." Frontiers in Chemistry, 2022, 10: 814880.
3. Vilella A, Ruozi B, Belletti D, et al. Endocytosis of nanomedicines: the case of glycopeptide engineered PLGA nanoparticles[J]. Pharmaceutics, 2015, 7(2): 74-89. https://doi.org/10.3390/pharmaceutics7020074.
4. Battogtokh G, Joo Y, Abuzar S M, et al. Gelatin coating for the improvement of stability and cell uptake of hydrophobic drug-containing liposomes[J]. Molecules, 2022, 27(3): 1041. https://doi.org/10.3390/molecules27031041.
5. Kashapov R, Ibragimova A, Pavlov R, et al. Nanocarriers for biomedicine: From lipid formulations to inorganic and hybrid nanoparticles[J]. International Journal of Molecular Sciences, 2021, 22(13): 7055. https://doi.org/10.3390/ijms22137055.