Extracellular vesicle-mediated protein delivery to the liver

Extracellular vesicles (EVs) are nanoscale particles that facilitate intercellular communication. They are regarded as a promising natural drug delivery system for transporting and delivering bioactive macromolecules to target cells. Recently, researchers have engineered EVs with FKBP12/FRB heterodimerization domains that interact with rapamycin to load and deliver exogenous proteins for both in vitro and in vivo applications. Researchers at the University Medical Center Utrecht examined the tissue distribution of EVs using near-infrared fluorescent imaging. The researchers evaluated the effectiveness of EV-mediated delivery of Cre recombinase specifically to hepatocytes in the livers of Ai9 Cre-loxP reporter mice. Intravenous injection resulted in more efficient Cre protein delivery to the liver than intraperitoneal injections. Depleting liver-resident macrophages with clodronate-encapsulated liposome pre-treatment did not enhance EV-mediated Cre delivery to hepatocytes. Moreover, they demonstrated that multiple intravenous injections of Cre-EVs facilitated functional Cre delivery to hepatocytes. To the best of their knowledge, this is the first study to simultaneously investigate the tissue distribution of FKBP12/FRB-engineered EVs and their subsequent intracellular protein delivery in Ai9 Cre-loxP reporter mice. These insights can inform preclinical research and contribute to developing next-generation EV-based platforms for delivering therapeutic proteins or genome editing technologies targeting the liver.

Higher liver protein delivery upon intravenous administration of Cre-EVs as compared to intraperitoneal injection

(a) Schematic illustration of labeling EVs with an Alexa Fluor NHS ester, followed by dye quenching and separating the free dye from the labeled EVs through SEC. (b) NTA analysis of AlexaFluor647-labeled EVs showed size distribution between ± 50–200 nm. The uptake of AlexaFluor647-labeled EVs (in red) was confirmed in (c) HepG2 and (d) NIH3T3 cells by fluorescent microscopic analysis. Representative live images of in vivo tracking of AlexaFluor790-labeled EVs in Ai9 Cre-loxP reporter mice that received Cre-EVs through (e) i.v.-or (f) i.p. injection over 120 h by NIRF imaging. (g) Schematic illustration of Cre-mediated recombination in Ai9 Cre-loxP reporter mice, wherein the functional intracellular delivery of Cre will lead to Cre-recombined tdTomato+ cells. (h) Immunofluorescence images from liver sections. Blue = Hoechst, Red = tdTomato+, scale bar = 50 μm. (i) Immunofluorescence quantification of total tdTomato+Hoechst+ cells in liver tissue sections from Ai9 Cre-loxP reporter mice that received Cre-EVs either through i.v. or i.p. injection. Statistical analysis was performed via an unpaired t-test with p-values *<0.05, **<0.01, ***<0.001, and ****<0.0001 considered statistically significant. Per group n = 3, total n = 6.

Ilahibaks NF, Roefs MT, Brans MAD, Blok CS, de Jager SCA, Schiffelers RM, Vader P, Lei Z, Sluijter JPG. (2023) Extracellular vesicle-mediated protein delivery to the liver. J Ext Biol [Epub ahead of print]. [article]

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