Immunoliposomes: Definition, Application and Binding Mode

What are immunoliposomes?

Immunoliposomes are liposomes modified with monoclonal antibodies (Mabs), antibody Fab fragments (Fab), single-chain antibodies (scFv), or ligands. By attaching antibodies on the surface of liposomes, target cells are recognized, and the targeting of liposomes is improved, so as to avoid adverse reactions caused by drug delivery to non-action sites. The presence of antibodies makes immunoliposomes highly specific to the target, which can effectively improve the sensitivity of the analysis. After entering the human body, ordinary liposomes can be removed by the reticuloendothelial system and cannot reach the target area. Immunoliposomes are elongated-chain polyethylene glycol (PEG) on membranes, which can shield the recognition and clearance of the reticuloendothelial system (RES), thereby achieving high localization and high targeting. This technology has shown great potential in cancer, autoimmune diseases, infectious diseases, vaccines, and molecular imaging.

Fig.1 Structure of immunoliposomes and their application in disease. (Eloy Josimar O., et al., 2017)

Table 1. Antibody conjugation service at BOC Sciences.

History of the development of immunoliposomes

Since its discovery by Bangham about 50 years ago, liposomes have become a very promising tool in drug delivery systems. The first liposomal drug formulation approved by the United States Food and Drug Administration (FDA) was Doxil in 1995. Since then, several liposomal drug delivery products have been developed and are now commercially available such as DaunoXome, DepoCyt, Myocet, Lipodox, and Marqibo. So far, the development of liposomes has gone through three generations:

First-generation immunoliposomes

Antibody is directly attached to the lipid membrane surface of the liposome for active targeting. However, due to its short half-life due to its easy recognition and clearance by the reticuloendothelial system (RES) upon entering the body, its clinical application is limited. The first immunoliposome prepared by Torchilin, by covalently conjugating to an anti-cardiac myosin antibody, showed in vitro antibody activity specific to all oradioactive iodine-labeled antigens.

Second-generation immunoliposomes

To extend the cycle time, the liposome surface is not only attached to the antibody fragment, but also modified with polyethylene glycol (PEG). However, the long PEG chain may shield the binding of the antibody to the antigen, reducing the targeting rate.

Third-generation immunoliposomes

Antibodies are attached to the ends of polyethylene glycol or its derivatives to make sterically stabilized immunoliposomes (SIL). This design prolongs the blood circulation time of drug-loaded liposomes, while retaining the targeted function of antibodies, with high stability and long-cycle characteristics. In addition, there are new liposomes such as prebody liposomes, membrane fusion liposomes, flexible liposomes, biomimetic liposomes, and compound immunoliposomes.

Structure and function of immunoliposomes

At the heart of the immunoliposome is a lipid bilayer with specific antibodies or antibody fragments attached to its outer surface. The presence of antibodies enables immunoliposomes to efficiently recognize and bind to target cells, improving the targeting and therapeutic effect of drugs.

Lipid bilayer: Composed of natural or synthetic phospholipid molecules, forming a hydrophilic core and hydrophobic shell. This bilayer encapsulates both hydrophilic and hydrophobic drugs, providing protection and controlling drug release.

Antibodies or antibody fragments: Chemically or physically conjugated to the surface of liposomes that enable liposomes to recognize specific cells or tissues. Commonly used antibody types include intact antibodies, monoclonal antibodies, antibody Fab fragments, and single-chain antibodies (scFv).

Polyethylene glycol (PEG): By modifying PEG on the surface of liposomes, it can increase its circulation time in the body and reduce the risk of being cleared by the immune system.

Fig.2 PEGylated doxorubicin immunoliposome surface. (Khan Amjad Ali, et al., 2020)

The binding mode of immunoliposomes

There are several ways to bind an antibody or its fragments to liposomes, with chemical conjugation of active functional groups being the most commonly used. Antibodies or antibody fragments are chemically conjugated to the liposome surface. Commonly used coupling methods include the formation of thioether bonds, disulfide bonds, amide bonds, etc.

Thioether bonds

Thioether bonding is a more commonly used coupling method, often using the maleimide group at the end of the phospholipid derivative material and the sulfhydrylated antibody through thioether bonding, or by reducing agent to the phospholipid-derived material containing pyridine disulfide propyl amide (PDP) to produce a free sulfhydryl group, and then with the antibody introduced into the maleimide group through the thioether bonding.

Disulfide bonds

Similar to thioether bond coupling, disulfide bond coupling is often directly linked to a sulfhydrylated antibody using a phospholipid-derived material containing PDP, and 2-thio-pyridone is removed to form a disulfide bond. Phospholipid-derived materials containing PDP can also be reduced with reducing agents to produce free sulfhydryl groups, which then form disulfide bonds with 3-(2-pyrimidine dithiol)propionate N-hydroxy succinimidyl ester-modified antibodies.

Amide bonds

Amide bond coupling does not require modification of the antibody, and directly uses the amino group on the antibody to react with the carboxyl group at the end of the coupling agent on the liposome to form an amide bond. Elbayoumi and Torchilin prepared an antinucleosome monoclonal antibody 2C5-mediated doxorubicin-loaded immunoliposome by reacting the amino group on the antinucleosome monoclonal antibody 2C5 antibody with the terminal carboxyl group of the phospholipid-derived material to form an amide bond and to actively target the treatment of tumor-bearing mice.

Non-covalent bonds

Antibodies can also bind to liposomes through non-covalent bonds, such as the preparation of immunoliposomes using the affinity of biotin-ovalbumin and nickel-polyhistidine.

Application of immunoliposomes

Immunoliposomes have demonstrated a wide range of applications in several medical fields:

Cancer treatment

Immunoliposomes can precisely deliver anti-cancer drugs to cancer lesions by specifically targeting tumor cells, enhancing the therapeutic effect and reducing side effects. Trastuzumab is a monoclonal antibody used to treat malignancies that express human epidermal growth factor receptor 2 (HER2). For example, Yang successfully conjugated thiolated trastuzumab to the end of a PEG to prepare paclitaxel-lab-loaded SIL for the treatment of HER2-positive breast cancer by post-insertion. Confocal microscopy showed that the immunoliposome could well target HER2 and significantly increase the drug concentration at the tumor site, achieving the purpose of treating breast cancer with high HER2 expression.

Biosensors and molecular imaging

By modifying fluorescent markers or other detection molecules on the liposome surface, immunoliposomes can be used for in vivo imaging and biosensor techniques to aid in early diagnosis and monitoring of disease. For example, Feng fused protein A with antibody affinity to luciferase and used the fusion protein to link an anti-EGFR monoclonal antibody to a liposome to prepare luciferase-tagged immunoliposomes. The liposome can not only target tumor cells with high expression of EGFR, but also realize the monitoring of the in vivo targeted action process of liposomes through the bioluminescence effect of luciferase, which has the dual potential of imaging monitoring and targeted therapy.

Targeted drug delivery systems

Immunoliposomes are liposomes modified with monoclonal antibodies or antibody fragments, which belong to actively targeted liposomes, which have complement and antibody-dependent synergistic cell killing effects, and because immunoliposomes have good targeting, they can avoid adverse reactions caused by drug delivery to non-action sites.

Summary

As a novel delivery system, immunoliposomes have great potential in multiple fields such as cancer therapy, gene therapy, and treatment of central nervous system diseases with large payload capacity, altered pharmacokinetics, improved drug tolerance, and enhanced lesion-specific distribution. By continuously optimizing its structure and preparation methods, immunoliposomes are expected to further improve the efficacy and safety of drugs and promote the development of modern medicine.