Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells

Abstract

RNA-based therapeutics hold great promise for treating diseases and lipid nanoparticles (LNPs) represent the most advanced platform for RNA delivery. However, the fate of the LNP-mRNA after endosome-engulfing and escape from the autophagy-lysosomal pathway remains unclear. To investigate this, mRNA (encoding human erythropoietin) was delivered to cells using LNPs, which shows, for the first time, a link between LNP-mRNA endocytosis and its packaging into extracellular vesicles (endo-EVs: secreted after the endocytosis of LNP-mRNA). Endosomal escape of LNP-mRNA is dependent on the molar ratio between ionizable lipids and mRNA nucleotides. Our results show that fractions of ionizable lipids and mRNA (1:1 molar ratio of hEPO mRNA nucleotides:ionizable lipids) of endocytosed LNPs were detected in endo-EVs. Importantly, these EVs can protect the exogenous mRNA during in vivo delivery to produce human protein in mice, detected in plasma and organs. Compared to LNPs, endo-EVs cause lower expression of inflammatory cytokines.

Biofabrication of Cell-Derived Nanovesicles: A Potential Alternative to Extracellular Vesicles for Regenerative Medicine

“…Importantly, we used donor cells stably expressing Cre, allowing us to study only cell-derived vesicle-mediated transfer of Cre cytosolic RNA (functional RNA) and protein, without the interference of lipid-based carriers that may end up in NVs/EVs and affect their behavior. [36][37][38][39].…”

Thirty Years of Cancer Nanomedicine: Success, Frustration, and Hope

“…Even if the real efficacy of active targeting in cancer affected patients is still in debate, this kind of approach allowed improvement in the awareness about the molecular characteristics of different cancer types [10,72,81]. Moreover, the evidence that NCs could be entrapped inside endosomes and/or lysosomes led the scientists to study this mechanism developing NCs able to reach the cell cytosol by clever strategies, including direct fusion with the plasma membrane [59] or by performing enhanced endosomal escape [82][83][84]. The use of engineered NCs can bring other opportunities, such as the encapsulation of poorly soluble drugs [85][86][87], as well as the delivery of biologics improving their bioavailability, permeability, and stability in the biological environment [53,72].…”

How does temperature play a role in the storage of extracellular vesicles?

“…They can (Valadi et al, 2007;Zomer et al, 2015). In addition to normal physiological functions, EVs also play an important role in the occurrence, development, diagnosis, and treatment of various diseases, including tumors (Mao et al, 2019;Maugeri et al, 2019;Morad et al, 2019;J. Wang et al, 2019), obesity (Ji & Guo, 2019), viral myocarditis (Li et al, 2019), diabetes (M. , Alzheimer's/Parkinson's disease (Grozdanov et al, 2019;Jia et al, 2019), and so forth (Table 1).…”