Lipid nanoparticles, or LNPs, are microscopic particles used for delivering therapeutic molecules into cells. A primary use for these particles is the delivery of genetic material, such as messenger RNA (mRNA), which can instruct cells to produce specific proteins. After an LNP is taken up by a cell, it is enclosed within a membrane-bound bubble called an endosome. For the therapeutic cargo to work, it must get out of this compartment and reach the cell’s interior, known as the cytoplasm.
This process is called endosomal escape. The efficiency of this escape is a major focus of research and development in nanomedicine, as trapped cargo can be destroyed by the cell.
The Cellular Journey and the Endosomal Barrier
When LNPs encounter a cell, they are taken in through endocytosis, where the cell membrane folds inward to form a vesicle called an endosome. This endosome is the starting point of a dynamic sorting pathway. It begins as an “early endosome,” a hub that determines the fate of its contents.
From here, materials can be recycled to the cell surface or trafficked deeper into the cell. If not rerouted, the early endosome matures into a “late endosome,” which has a more acidic internal environment.
The final destination for many substances in this pathway is the lysosome, an organelle filled with powerful enzymes that break down cellular waste. If an LNP remains within the endosome as it fuses with a lysosome, its genetic payload will be destroyed. This degradation pathway makes the endosome a barrier that must be overcome for successful drug delivery.
The Need for Cytoplasmic Access
Endosomal escape is necessary because of the nature of the therapeutic cargo. Genetic materials like mRNA and small interfering RNA (siRNA) cannot function while inside an endosome. These molecules must reach the cytoplasm, the substance that fills the cell, to interact with the cellular machinery.
For an mRNA therapeutic to be effective, it needs to be translated into a protein by ribosomes located in the cytoplasm. Similarly, siRNA silences specific genes by interacting with the RNA-induced silencing complex (RISC), which also operates in the cytoplasm.
Researchers have found that while cells may take up many LNPs, a small fraction, sometimes less than 2%, of the cargo successfully escapes. This low efficiency highlights the challenge in designing LNPs that can reliably free their contents.
Mechanisms of LNP-Mediated Endosomal Escape
Scientists believe LNPs employ several mechanisms to break out of the endosome. A prominent theory is the “proton sponge” effect. As the endosome matures, its interior becomes more acidic. Ionizable lipids within the LNP become positively charged by absorbing protons. This influx of protons prompts the cell to pump in even more protons and negatively charged chloride ions to balance the charge, leading to an increase in osmotic pressure that causes the endosome to swell and eventually rupture, releasing its contents.
Another proposed mechanism involves direct interaction with the endosomal membrane. The lipids making up the LNP can be designed to encourage fusion with the endosome’s membrane. In this process, the two membranes merge, creating an opening for the cargo to pass into the cytoplasm.
Some research suggests certain LNP structures might form transient pores in the endosomal membrane. These pores would be large enough for the payload to slip through without a complete rupture of the vesicle. The specific internal structure of the LNP, such as whether it has a more ordered or disordered arrangement of lipids, can influence which of these escape mechanisms is more likely to occur.
Factors Modulating Escape Efficiency
The efficiency of endosomal escape is influenced by factors related to the LNP and the target cell. The LNP’s composition is a primary determinant. The types and ratios of lipids used, such as ionizable lipids and cholesterol, can alter escape capabilities. The pKa of an ionizable lipid—the pH at which it becomes charged—is a finely tuned property. A well-designed lipid remains neutral in the bloodstream but becomes protonated in the acidic endosome to initiate escape.
The physical properties of the nanoparticles also play a role. Factors like the size, surface charge, and shape of the LNP affect how it interacts with the endosomal membrane. Research has shown that LNPs with certain non-lamellar, or less layered, internal structures are better able to release their cargo. These structural details can be influenced by the manufacturing process and the interaction between the lipids and the RNA cargo.
Finally, cellular factors introduce another layer of variability. Different cell types can have endosomes with varying acidity or membrane compositions. The rate at which endosomes mature can also differ, creating a narrower or wider window for escape.