Development of Fine Liposome Nanoplatforms for Macrophage Clearance

Although the precise composition of the tumor microenvironment (TME) varies in an organ-dependent manner, tumor-associated macrophages (TAMs) have emerged as one of the most numerous and critical cell types, with potent multifaceted roles in regulating cancer progression and modulating the response to therapeutic intervention. By decreasing the permissive nature of the tumor microenvironment, macrophage depletion may allow the host's defenses to recover and eliminate tumor cells.

The researchers developed four types of click chemistry-based liposome nanoplatforms that encapsulate clodronate, an active pharmaceutical ingredient in the treatment of inflammatory diseases such as rheumatoid arthritis and osteoarthritis, into uniformly sized liposomes, which are then conjugated with and radiolabeled with Man-N3. Functionalization of Man-N3 improves specific targeting of M2 macrophages, and radioisotope labeling enables the in vivo imaging of the liposome nanoplatforms.

Fig. 1 Development of finely tuned liposome nanoplatform for macrophage depletion.¹

By assembling top-class scientific experts and integrating a state-of-the-art macrophage therapeutics development platform as well as industry-leading expertise in the depletion of TAM, Creative Biolabs offers accurate and effective solutions for our clients.

Our Services	Descriptions
Tumor-associated Macrophage (TAM) Depletion Service	With an experienced team of in-house TAM depletion experts, Creative Biolabs employs efficient strategies, including depletion of TAMs by colony-stimulating factor 1 receptor (CSF1R) blockade and specific depletion of TAMs by mAbs, to provide our clients with highly customizable solutions.
Macrophage-targeted Drug Delivery System Development	Based on the unrivaled expertise and experiences in immunotherapy discovery and development, Macrophage-targeted drug delivery system development services at Creative Biolabs include but are not limited to nanoparticle drug delivery system, liposome drug delivery system, dendrimer drug delivery system.

Liposome Nanoplatforms for Macrophage Clearance

The researchers synthesized a 100 nm liposome nanoplatform. Four types of macrophage-targeted liposomes, including liposomes, mannosylated liposomes, chlorophosphonate-encapsulated liposomes, and chlorophosphonate-encapsulated mannosylated liposomes. The liposome nanoplatforms were observed by transmission electron microscopy (TEM) to be spherical with uniform size distribution.

After phagocytosis of clodronate liposomes by macrophages, the phospholipid bilayer was disrupted by lysosomal phospholipases. Subsequently, clodronate is released intracellularly, leading to cell death through irreversible functional damage and apoptosis. Clodronate mannosyl liposomes effectively reduced M2 macrophages in normal liver and tumor microenvironments in vitro.

The results showed that this liposome nanoplatform has fine particle size control, high in vivo stability and excellent in vitro M2 macrophage targeting and depletion, making it a promising macrophage depleting agent.

Evaluation of Liposome Nanoplatforms

In the study, the researchers evaluated the macrophage depletion effect of liposome nanoplatforms. The following assessment scheme was primarily used.

Assessment Methods	Objective	Results
In Vitro Cell Viability Testing	To determine toxicity at the cellular level	The clodronate-free liposomes did not alter cell viability compared to the control. This indicates that the synthesized liposomes are reasonably biocompatible. The clodronate liposomes decreased cell viability in a dose-dependent manner.
Effect on Macrophage Uptake In Vitro	To assess the degree of specific binding to macrophages	Images were taken to observe the cellular uptake of liposome nanoplatforms. The fluorescence signal of mannosylated liposomes was significantly higher than that of mannose-free liposomes. The nanoplatform specifically binds to cells by targeting the ligand mannose.
*Pharmacokinetics and In Vitro* Biodistribution**	To determine the changes in their distribution over time	Biodistribution studies of liposome nanoplatforms were performed using positron emission tomography (PET) imaging. The biodistribution of liposomes after clodronate treatment was quite different. Clodronate-encapsulated liposomes accumulated in the liver and spleen. This may be due to the fact that the liver and spleen are sensitive to blood vessels and contain several types of tissue macrophages.
Immunofunctional Evaluation of Macrophage Clearance from Hepatic Tissues In Vitro	To determine whether liposomes are effective in reducing macrophages in liver tissue	Clodronate-encapsulated mannosylated liposomes showed the highest depletion of liver macrophages. It enters M2 macrophages via lattice protein-mediated endocytosis, which is effectively internalized via surface overexpression of the mannose receptor. This induces clodronate-mediated apoptosis, leading to M2 macrophage depletion. Liposomes are not hepatotoxic.
Evaluation of Efficacy on Macrophage Clearance by TME In Vitro	To confirm the efficacy of liposome nanoplatforms for TME macrophage depletion	Clodronate-encapsulated mannosylated liposomes most effectively blocked tumor progression. Clodronate-encapsulated mannosylated liposomes effectively achieved specific targeting of M2 macrophages in TAMs in TME and induced macrophage depletion by apoptosis induced by CML-encapsulated sodium chloride.

As a result, this liposome nanoplatform has

Finely tuned size control.
High in vivo stability.
Excellent in vitro M2 macrophage targeting and depletion effects.

Tailoring Liposome Properties for Macrophage Clearance

Rational design of liposomal formulations can help optimize their interaction with macrophages while minimizing off-target effects. Key considerations include the choice of lipid composition, particle size, surface charge and surface functionalization strategies.

Lipid composition
By carefully selecting lipids with specific physicochemical properties, researchers can modulate membrane fluidity, stability and drug encapsulation efficiency. For macrophage targeting applications, lipids that mimic the composition of cell membranes are often used to enhance biocompatibility and reduce immunogenicity.
Particle size
Fine liposome nanoplatforms are carefully engineered to precisely control particle size, which typically ranges from tens to hundreds of nanometers. For macrophage clearance, smaller liposomes (<200 nm) exhibit enhanced cellular uptake and intracellular transport, facilitating efficient drug delivery to macrophages in various tissues and organs.
Surface charge
Negatively charged liposomes tend to exhibit prolonged blood circulation times due to the conditioning effects and reduced clearance by the reticuloendothelial system (RES). Conversely, positively charged liposomes can promote cellular uptake through electrostatic interactions with negatively charged cell membranes. By modulating surface charge, fine liposome nanoplatforms can be tailored to optimize macrophage targeting and clearance kinetics.
Surface functionalization
Various ligands, such as antibodies, peptides and small molecules, can be conjugated to the liposome surface to facilitate macrophage-mediated receptor-mediated uptake. Innovative bioconjugation techniques can be employed to precisely attach the targeted fraction while maintaining liposome stability and biocompatibility. This enables precise control of ligand density and orientation, ensuring optimal macrophage recognition and internalization.

In addition to fine-tuning liposome properties, it is possible to combine the benefits of liposomes with other nanoparticle systems. These hybrid systems offer unprecedented versatility and multi-functionality, opening up new avenues for synergistic approaches to macrophage-targeted therapies.

Reference

Choi, Tae Hyeon, et al. "Development of finely tuned liposome nanoplatform for macrophage depletion." Journal of Nanobiotechnology 22.1 (2024): 83.