PEGylated Liposomes in Clinical Trials

PEGylated liposomes are a nanocarrier system that covalently binds polyethylene glycol (PEG) molecules to the surface of liposomes. Modifying the PEG molecules to the surface of liposomes can significantly improve the stability of liposomes in the body. PEGylated liposomes have broad clinical application prospects. They can not only improve the bioavailability and safety of drugs, but also reduce the frequency of drug administration, thereby improving patient compliance. Therefore, PEGylation technology plays an important role in modern drug delivery systems.

What are Liposomes?

Liposomes are spherical vesicles composed of one or more phospholipid bilayers, encapsulating an aqueous core. This structure mimics biological membranes, making liposomes an ideal vehicle for drug delivery applications. The unique components of liposomes can encapsulate hydrophilic and hydrophobic molecules, thereby significantly improving the bioavailability, stability and targeted delivery of therapeutic agents. At present, liposomes have been extensively studied in the clinical environment because of their ability to improve the therapeutic index of drugs, especially in the field of oncology. They can improve the delivery of tumor sites and reduce systemic toxicity. Liposomes have several advantages in drug delivery:

Fig. 1. Liposomal structure (Heliyon. 2022, 8(5): e09394).

• Biocompatibility: Liposomes are made from naturally occurring phospholipids, ensuring minimal toxicity and high biocompatibility with human tissues.
• Versatility: They can encapsulate a wide range of substances, including small molecules, proteins, and nucleic acids, providing a versatile platform for drug delivery.
• Targeted Delivery: By modifying the liposome surface, specific targeting can be achieved, directing drugs to particular tissues or cells and reducing off-target effects.
• Controlled Release: Liposomes can be engineered to release their contents in a controlled manner, improving the therapeutic efficacy and reducing side effects.

What is PEGylation?

PEGylation is the process of attaching a polyethylene glycol chain to a molecule or surface (such as a protein, lipid, or drug delivery system) to improve its pharmacokinetic and pharmacodynamic properties. This modification significantly improves the solubility, stability and half-life of the therapeutic agent in the blood by reducing the recognition and clearance of the therapeutic agent by the immune system. In liposomes, PEGylation involves incorporating PEG-lipids into the liposome bilayer to form a hydrophilic 'invisible' coating, thereby minimizing protein adsorption and conditioning. This allows liposomes to avoid the mononuclear phagocyte system (MPS), thereby prolonging circulation time and improving accumulation in target sites such as tumors by enhancing permeability and retention (EPR) effects.

PEGylated Liposomes

PEGylated liposomes are a class of liposomal formulations in which polyethylene glycol chains are attached to the lipid bilayer surface. This modification enhances the ability of the liposomes to circulate in the bloodstream for extended periods of time, reduces their uptake by the reticuloendothelial system (RES), and increases their accumulation at target sites. Currently, PEGylated liposomes have been explored in numerous clinical trials for a variety of therapeutic areas, including oncology, infectious diseases, and genetic diseases. Their ability to encapsulate a wide range of therapeutic agents, from small molecules to nucleic acids, makes them a versatile platform for drug development. The key properties of PEGylated liposomes include:

• Steric stability: The presence of polyethylene glycol creates a steric barrier that prevents aggregation and fusion of liposomes, which is essential for maintaining the integrity of the formulation in circulation.
• Extended circulation time: By evading the immune system, PEGylated liposomes can maintain therapeutic levels of drugs for longer periods of time, which is particularly beneficial for chronic disease and cancer treatment.
• Passive targeting: The enhanced permeability and retention (EPR) effect observed in tumor tissues enables PEGylated liposomes to passively accumulate at tumor sites, thereby improving the therapeutic index of anticancer agents.

PEGylated Liposomes from BOC Sciences

Marketed PEGylated Liposomal Products

Several PEGylated liposomal formulations have successfully transitioned from clinical trials to the market, demonstrating their clinical efficacy and safety. These products highlight the therapeutic potential of PEGylated liposomes, particularly in oncology, where enhanced drug delivery and reduced toxicity are critical for treatment success. Below is a summary of key marketed PEGylated liposomal products:

PEGylated Liposomes in Clinical Trials

The clinical impact of PEGylated liposomal products continues to expand as more formulations enter clinical trials and are approved for therapeutic use. Their ability to improve pharmacokinetics, reduce toxicity, and enhance drug targeting makes them an essential tool in modern medicine, with the potential to significantly improve patient outcomes across various diseases. Continued advancements in PEGylation technology and liposome design promise to broaden the scope of clinical applications, from cancer and infectious diseases to gene therapy and immunotherapy.

PEGylated Liposomes for Oncology

In oncology, PEGylated liposomal formulations have revolutionized the treatment of several cancers. Drugs like Doxil® (PEGylated liposomal doxorubicin) have become standard treatments for cancers such as ovarian cancer, multiple myeloma, and Kaposi's sarcoma. The PEGylation process prolongs the circulation of the liposomes, allowing the encapsulated drug to accumulate in tumor tissues through the EPR effect. This mechanism reduces the systemic toxicity typically associated with chemotherapeutic agents, improving the therapeutic index and patient outcomes by minimizing damage to healthy tissues. Additionally, PEGylated liposomal products offer greater drug stability, protecting the active ingredient from degradation before reaching the target site.

PEGylated Liposomes for Infectious Diseases

Beyond oncology, PEGylated liposomal products are also finding significant use in the treatment of infectious diseases. Ambisome® (PEGylated liposomal amphotericin B) is a notable example used to treat severe fungal infections such as invasive aspergillosis and cryptococcal meningitis. The PEGylated liposomes protect the drug from rapid clearance by the MPS and enhance drug delivery to infected tissues. The reduced toxicity of amphotericin B in its PEGylated liposomal form allows for higher dosing and more effective treatment of life-threatening infections.

PEGylated Liposomes for Gene Therapy

In the field of gene therapy, PEGylated liposomes are being explored for the delivery of genetic material such as plasmids, siRNA, and mRNA. By encapsulating nucleic acids within PEGylated liposomes, researchers aim to overcome the challenges of instability, rapid clearance, and poor cellular uptake. These delivery systems enhance transfection efficiency, making them a promising tool for treating genetic disorders, cancers, and viral infections.

PEGylated Liposomes for Immunotherapy

PEGylated liposomes have also been investigated for use in immunotherapy, where they serve as carriers for immunomodulatory agents, vaccines, or antibodies. Their ability to modulate immune responses while targeting specific tissues has opened new avenues in treating autoimmune diseases, chronic inflammation, and for enhancing vaccine efficacy in infectious disease prevention.

PEGylated Liposomes Preparation

PEGylation can be performed in two ways: adding PEG-lipid to the lipid composition before liposome formation (pre-insertion method) or mixing PEG-lipid with liposome dispersion (post-insertion method). Both PEG length and coverage density affect the efficiency of PEGylation of liposomes. Very short PEG molecules cannot prevent protein absorption and increase blood circulation time, while very long PEG chains lead to a significant decrease in transfection activity. Generally, medium-length PEG molecules are used for liposome modification. The higher the molar PEG-lipid/lipid composition ratio, the higher the coverage density (Fig. 2):

• <5% PEG produces less than 100% coverage, mushroom-like;
• 5-15% PEG produces ∼100% coverage, mushroom or brush-like;
• >15% PEG produces ∼100% coverage, brush-like.

Fig. 2. PEGylated liposomes (Medchemcomm. 2019, 10(3): 369-377).

Pre-insertion PEGylation

The pre-insertion method is traditionally employed for the preparation of PEGylated liposomes. In this technique, a thin lipid film containing a mixture of standard lipids and PEG-lipids is formed by evaporating organic solvents under reduced pressure. The resulting lipid film is then hydrated with an appropriate aqueous buffer, often aided by sonication to ensure uniform dispersion. For instance, phosphatidylethanolamines (PE) such as DSPE-mPEG, DPPE-mPEG, and DOPE-mPEG are commonly used. These lipids differ in their hydrophobic domains, PEG chain lengths, and fatty acid compositions, providing a range of properties suitable for various applications. After hydration, nucleic acids (e.g., plasmid DNA or siRNA) can be added to form PEGylated lipoplexes, allowing for efficient delivery into target cells. Notably, studies have demonstrated that liposomes modified with PEG 2000 are significantly more effective in targeting tumor tissues compared to their non-modified counterparts, highlighting the importance of PEG in enhancing therapeutic outcomes.

Post-insertion PEGylation

On the other hand, the post-insertion method offers a different approach to achieve PEGylation. This method is typically applied after the drug loading step, wherein PEG-lipid solutions or micelles are slowly introduced into the pre-formed liposomal dispersion. A significant advantage of this technique is its ability to achieve a higher PEG coverage, often exceeding 5%. The interaction between PEG derivatives and specific hydrophobic anchors in the liposomal membrane ensures that PEGylated conjugates are incorporated solely into the outer bilayer. For example, mPEG-HDAS (a PEG derivative) can inhibit complement system activation, thus enhancing the stability and circulation time of the liposomes in the bloodstream.

The choice between pre-insertion and post-insertion methods often hinges on the desired characteristics of the final product. Pre-insertion is well-suited for applications where low PEG motif density is acceptable, while post-insertion is preferred when a higher level of PEGylation is necessary. Furthermore, post-insertion has been shown to preserve the conventional drug loading techniques without compromising encapsulation efficiency. The method allows for the use of PEGylated ceramides, which have demonstrated improved transfection efficiencies in liposome/siRNA complexes compared to pre-insertion methods, due to the formation of a hexagonal phase that enhances membrane fusion with target cells.

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References

1. Nsairat, H. et al. Liposomes: structure, composition, types, and clinical applications. Heliyon. 2022, 8(5): e09394.
2. Nosova, A.S. et al. Diversity of PEGylation methods of liposomes and their influence on RNA delivery. Medchemcomm. 2019, 10(3): 369-377.