Endosomal escape: A bottleneck for LNP-mediated therapeutics

Abstract

Lipid nanoparticles (LNPs) have recently emerged as a powerful and versatile clinically approved platform for nucleic acid delivery, specifically for mRNA vaccines. A major bottleneck in the field is the release of mRNA-LNPs from the endosomal pathways into the cytosol of cells where they can execute their encoded functions. The data regarding the mechanism of these endosomal escape processes are limited and contradicting. Despite extensive research, there is no consensus regarding the compartment of escape, the cause of the inefficient escape and are currently lacking a robust method to detect the escape. Here, we review the currently known mechanisms of endosomal escape and the available methods to study this process. We critically discuss the limitations and challenges of these methods and the possibilities to overcome these challenges. We propose that the development of currently lacking robust, quantitative high-throughput techniques to study endosomal escape is timely and essential. A better understanding of this process will enable better RNA-LNP designs with improved efficiency to unlock new therapeutic modalities.

Keywords: LNPs; RNA vaccines and therapeutics; endo-lysosomes; endosomal escape; mRNA.

Fig. 1.

Schematic representation of the bilayer to hexagonal phase transition of LNPs. Under the acidic pH of endolysosomal compartment, ionizable lipids are positively charged and interact with the anionic lipids present on the inner leaflet of the endosomal membrane. This interaction leads to the transition from bilayer to hexagonal phase transition resulting in endosomal membrane damage and cargo release.

Fig. 2.

Schematic representation of the Proton Sponge Effect: Due to the buffering ability of ionizable lipids, there is a huge influx of protons by activation of the proton pump in endolysosomal compartments. To neutralize the membrane potential, an inflow of chloride ion is triggered creating an osmotic imbalance which is followed by water intake. This leads to endolysosomal compartment swelling and eventually burst, which releases the cargo.

Fig. 3.

Schematic representation of endocytosis mechanisms. Figure showing LNP uptake by clathrin-mediated endocytosis and macropinocytosis. Once inside the cell, particles are transferred to early endosomes. From this stage, there are various reports on exact endosomal compartment of escape. Various reports claim that the cargo is released from a hybrid compartment having the properties of early as well as late endosome (17, 18), the late endosomal compartment (20), and RAB11 +ve recycling endosomes (23).