Role of Endosomal Escape in Lipid Nanoparticle for Drug Delivery

One of the most promising delivery platforms for drugs are LNPs, especially in RNA therapy such as mRNA vaccines. But for all their popularity, the successful transfer of genetic material enclosed in LNPs is restricted by a huge barrier - endosomal escape. This mechanism by which LNPs transport their payload into the cytoplasm is still one of the toughest problems.

What is an Endosome?

Endosomes are membrane-bound structures inside of eukaryotic cells that develop during endocytosis. Endocytosis involves the entry into the cell of extracellular materials contained in vesicles. These vesicles turn into early endosomes, which go on to mature through the maturation process and become late endosomes. They are key for sorting, trafficking and recycling cellular waste. Such vesicles are essential for cellular capture of nanoparticles, such as those that deliver RNA. The dynamic nature of endosomes makes it possible to compartmentalise and modulate molecules such as nutrients, growth factors and, in the case of LNPs, therapeutics. But they have an acidic pore and membrane structure that make it difficult for LNP cargo to be efficiently delivered into the cytoplasm.

Endosomal Escape

Endosomal escape is a crucial process in the delivery of therapeutic agents, particularly when using nanocarriers like lipid nanoparticles (LNPs), viral vectors, or polymer-based systems. This mechanism enables the cargo encapsulated within endocytic vesicles to reach its intended intracellular target, typically the cytoplasm or the nucleus. Without successful endosomal escape, therapeutic molecules are often trapped in the endosome or degraded in lysosomes, hindering their therapeutic effects. Endocytosis is the cellular process by which external substances, including nanoparticles or other drug delivery vehicles, are internalized into the cell. Once inside the cell, the material is encapsulated in an endosome—a membrane-bound vesicle that functions as an intermediate compartment in the cellular trafficking system. Endosomes play a critical role in sorting and directing internalized cargo to various destinations, including recycling, degradation, or translocation to other cellular compartments. However, the cargo within endosomes must escape to effectively exert its therapeutic action. The endosomal membrane acts as a physical barrier, preventing the cargo from interacting with the cytoplasm. If escape does not occur, the contents of the endosome are often degraded in the lysosomes, rendering the therapeutic agents ineffective. Thus, endosomal escape is the pivotal event that determines the success of many drug delivery systems, particularly in the context of gene therapies and RNA-based therapeutics.

Schematic representation of the endosomal escape mechanism for carrier delivery of therapeutic nucleic acids. ()

Endosomal Escape LNP

The delivery of therapeutic nucleic acids, including mRNA, requires the endosomal escape of LNPs into the cytoplasm. If endosomal escape fails to occur, the LNPs and their cargo are often degraded in the lysosomes, rendering the treatment ineffective. Therefore, the efficiency of this step is pivotal for the success of LNP-based delivery systems. Lipid nanoparticles (LNPs) are typically composed of ionizable lipids, cholesterol, phospholipids, and polyethylene glycol (PEG)-lipid conjugates. These components are carefully designed to optimize stability, solubility, and efficient cellular uptake. However, despite their sophisticated design, endosomal escape remains a limiting factor for effective LNP delivery. The ionizable lipid component of LNPs plays a critical role in facilitating endosomal escape. Upon internalization, these lipids become protonated in the acidic endosomal environment, which leads to membrane destabilization and enhanced release of the LNP cargo. The presence of PEG-lipids in LNP formulations helps to stabilize the particle and avoid aggregation, further promoting efficient delivery.

Endosomal Escape Mechanism of Lipid Nanoparticles

Several mechanisms have been proposed to explain how lipid nanoparticles manage to escape from endosomes:

pH-Dependent Membrane Disruption

One of the most studied mechanisms for endosomal escape is the pH-dependent disruption of the endosomal membrane. Endosomes mature into acidic compartments, which lower the pH within the vesicle to around 5. This acidic environment triggers the destabilization of the LNP's lipid bilayer. As the pH drops, the protonation of amines in the LNPs' lipid components can lead to membrane disruption, enabling the release of the encapsulated cargo into the cytoplasm. Many LNP formulations are designed to exploit this pH-sensitive behavior for controlled escape.

Lipid-Mediated Fusion

Lipid-mediated fusion between the LNP membrane and the endosomal membrane is another proposed escape mechanism. The lipid bilayers of LNPs and endosomes are structurally similar, and under certain conditions, fusion can occur, leading to the formation of a hybrid structure. This fusion allows for the release of the LNP cargo into the cytoplasm. This approach is often facilitated by the presence of specific lipid components in the LNP formulation, such as ionizable lipids, that promote fusion.

Membrane Destabilization via Cationic Lipids

Cationic lipids, which are often used in the formulation of LNPs, are positively charged and can interact with the negatively charged endosomal membrane. This interaction can cause the membrane to destabilize, leading to rupture and cargo release. The cationic lipids play a crucial role in enhancing the ability of LNPs to escape the endosome by disrupting the integrity of the endosomal membrane.

Endosomal Escape via Membrane Phase Transition

Some LNPs utilize a phase transition mechanism, where the temperature or pH-induced changes in the lipid phase lead to destabilization of the endosomal membrane. In this scenario, the lipid molecules within the LNP transition from a gel phase to a liquid crystalline phase, making it easier for the LNPs to integrate with the endosomal membrane and facilitate escape.

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How to Enhance Endosomal Escape?

Improving endosomal escape is crucial for the success of drug delivery systems, especially in gene therapies, RNA vaccines, and RNA-based treatments. Overcoming the endosomal barrier remains a key challenge, and several strategies are being developed to enhance the release of cargo from endosomes.

pH-Sensitive Nanomaterials

Nanomaterials that respond to the acidic pH inside the endosome (pH: 5.0–6.0) are a key strategy. Ionizable lipids or pH-sensitive polymers change their structure in low pH environments, disrupting the endosomal membrane and facilitating cargo release.

• Example: Ionizable lipids used in mRNA vaccines enable endosomal escape by becoming protonated in acidic conditions, destabilizing the endosomal membrane.

Cationic Lipids and Polymers

Cationic lipids and polymers interact electrostatically with the negatively charged endosomal membrane, promoting membrane fusion or disruption. This mechanism is particularly useful for gene delivery.

• Example: Cationic liposomes are used to deliver nucleic acids, promoting membrane fusion and enhancing cargo release.

Endosomal Membrane Fusion

Membrane fusion can be induced by incorporating fusogenic agents, such as fusogenic lipids (e.g., DOPE) or pH-sensitive peptides. These agents facilitate fusion between the nanoparticle and the endosomal membrane.

• Example: Liposomes containing fusogenic lipids undergo membrane fusion when exposed to acidic conditions, enabling cargo release.

Surface Modification of Nanoparticles

Surface modification enhances endosomal escape by replacing or supplementing PEGylation with other functional groups that promote membrane disruption or fusion, like cationic or amphiphilic molecules.

• Example: Nanoparticles with pH-sensitive PEG derivatives can destabilize the endosomal membrane at low pH, aiding in cargo release.

Osmotic Pressure and Swelling

Osmotic pressure buildup due to water influx into the endosome causes swelling, leading to membrane rupture and cargo release. This strategy is effective when combined with other approaches like pH-sensitive materials.

• Example: Polymeric nanoparticles that generate osmotic pressure upon endosomal uptake have shown enhanced cargo release.

Light and Ultrasound Triggered Release

External triggers, such as light or ultrasound, can induce conformational changes in nanoparticles that disrupt the endosomal membrane. These methods provide precise control over cargo release.

• Light-activated nanoparticles absorb light and undergo structural changes that destabilize the endosome.
• Ultrasound induces mechanical stress, causing endosomal rupture.

Exploiting Natural Endosomal Escape Mechanisms

Certain viruses, like adenoviruses, utilize proteins that promote endosomal escape. These proteins can be used to enhance nanoparticle delivery.

• Example: TAT peptides, derived from HIV-1, interact with endosomal membranes, aiding in the release of the encapsulated cargo.

Co-Delivery of Escape Agents

Co-delivery of additional agents that promote endosomal escape alongside the therapeutic cargo can increase overall delivery efficiency. These agents can include fusogenic peptides or cationic lipids.

• Example: Nucleic acid delivery systems often combine cationic lipids with membrane-disrupting peptides to enhance endosomal escape.

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Endosomal Escape Assay

Assessing the efficiency of endosomal escape is vital for the optimization of LNP formulations. Various assays are employed to measure the ability of LNPs to escape from endosomes and deliver their payload to the cytoplasm. These assays often involve tracking the fluorescence or other labels associated with the LNP cargo.

Fluorescence Microscopy

Fluorescence microscopy is a widely used technique for monitoring the cellular uptake of LNPs and their subsequent escape from endosomes. By labeling the LNPs with a fluorophore or using fluorescent markers for endosomal compartments, researchers can directly observe the localization and release of the LNP cargo in real time.

Flow Cytometry

Flow cytometry can be used to quantify the uptake of LNPs and the efficiency of their escape. This method allows for the measurement of fluorescence intensity within individual cells, providing insight into the cellular distribution of the nanoparticle cargo and the success of the escape process.

Confocal Microscopy

Confocal microscopy is another advanced technique that provides high-resolution images of the cellular localization of LNPs. By utilizing specific dyes or tags that fluoresce under certain wavelengths, researchers can study the process of endosomal escape in live cells, enhancing our understanding of the escape mechanisms.

Endosome vs Lysosome

Endosomes and lysosomes are both essential components of the cellular trafficking system, yet they serve distinct roles in cellular processes. Endosomes are involved in the sorting and internalization of extracellular materials, while lysosomes are primarily responsible for degrading and recycling cellular debris. While endosomes are typically acidic and involved in trafficking materials within the cell, lysosomes are highly acidic organelles that contain enzymes responsible for breaking down complex molecules. The transition from endosome to lysosome occurs when late endosomes fuse with lysosomes, leading to the degradation of the internalized contents. This fusion process is a major challenge for drug delivery systems, as encapsulated materials are often degraded in the lysosomes before they can reach their intended cellular targets.

What are Lysosomes?

Lysosomes are membrane-bound organelles that contain a variety of hydrolytic enzymes, such as proteases, lipases, and nucleases, capable of breaking down various biomolecules. Lysosomes play a critical role in cellular homeostasis by digesting and recycling cellular waste and foreign materials. They are often referred to as the "garbage disposal" of the cell. In drug delivery, lysosomes represent a significant barrier for the effective release of encapsulated materials. Once endosomes fuse with lysosomes, the acidic environment and enzymatic activity of lysosomes can rapidly degrade the cargo, preventing its intended therapeutic effect.

Why do Endosomes Fuse with Lysosomes?

Endosomes fuse with lysosomes as part of the process of material degradation and recycling within the cell. After endocytosis, the internalized material is sorted within early endosomes and transported through the maturation process, eventually becoming late endosomes. When the late endosomes mature, they fuse with lysosomes, and the contents are degraded by the hydrolytic enzymes present within the lysosomal lumen. This fusion process is vital for cellular regulation and the turnover of extracellular materials. However, for therapeutic drug delivery, this fusion is problematic, as it results in the degradation of the drug or genetic material encapsulated within nanoparticles. Therefore, preventing premature fusion with lysosomes and enhancing endosomal escape are key goals in the design of LNPs for therapeutic applications.

Endosomal escape remains one of the most significant hurdles in the effective delivery of therapeutic agents using lipid nanoparticles. Understanding the mechanisms behind endosomal escape, as well as the role of endosomes and lysosomes, is crucial for the development of more efficient drug delivery systems. By optimizing the design of LNPs and leveraging advanced assays to monitor escape efficiency, progress can be made toward overcoming this bottleneck. With further research and innovation, the therapeutic potential of LNP-based delivery systems will continue to expand, providing new opportunities for the treatment of a wide range of diseases.

Reference

1. Grau, M; et al. Strategies and mechanisms for endosomal escape of therapeutic nucleic acids. Current Opinion in Chemical Biology. 2024, 81: 102506.