Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis

Abstract

Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained.

Figure 1. Schematic representation of the intracellular delivery of EVs with the modification of arginine-rich cell-penetrating peptide for the active induction of macropinocytosis.

Objective therapeutic molecules are encapsulated in EVs by electroporation. EVs are then modified with stearyl-r8 peptide on EV membranes, resulting in the active induction of macropinocytosis and effective cellular uptake.

Figure 2. Increased cellular uptake of EVs by modification with stearyl-r8 peptide.

(a) TEM observation of CD63-GFP EVs (10 μg/ml) modified with stearyl-r8 (16 μM). (b) Confocal microscopic observation of HeLa cells treated with CD63-GFP EVs (10 μg/ml) modified with stearyl-r8 (16 μM) or r8 (16 μM) for 24 h at 37 °C (blue: Hoechst 33342; green: CD63-GFP EVs). (c) Relative cellular uptake of CD63-GFP EVs (10 μg/ml) modified with stearyl-r8 (16 μM) or r8 (16 μM) analyzed using a flow cytometer under the same experimental conditions as in (b). The data are expressed as the average (±SD) of three experiments. ^***p < 0.001.

Figure 3. Active induction of macropinocytosis by the modification of stearyl-r8 on the EV membrane.

(a) Relative cellular uptake of the macropinocytosis marker FITC-dextran in the presence or absence of EVs (10 μg/ml) with or without the modification of stearyl-r8 (16 μM) for 24 h at 37 °C analyzed using a flow cytometer. The data are expressed as the average (±SD) of three experiments. ^***p < 0.001. (b) Confocal microscopic image of HeLa cells treated with CD63-GFP EVs (10 μg/ml) modified with stearyl-r8 (16 μM) and the macropinocytosis marker Texas red-dextran for 24 h at 37 °C (green: CD63-GFP EVs; red: Texas red-dextran). “N” shows the nucleus. The arrows show the representative colocalization of EV and dextran. (c) Confocal microscopic image of HeLa cells treated with CD63-GFP EVs (10 μg/ml) modified with stearyl-r8 (16 μM) or EV without the peptide modification for 20 min at 37 °C. Cellular staining with rhodamine-phalloidin was performed to visualize F-actin prior to observation. The original images are shown in Supplementary Fig. 9. The arrows show representative lamellipodia formations.

Figure 4. Enhanced biological activity of saporin encapsulated in EVs by the modification of stearyl-r8.

(a) Schematic representation of the encapsulation of saporin (SAP) (PDB (Protein Data Bank) accession number: 1QI7) in EVs via electroporation. (b) Microscopic images of HeLa cells treated with saporin-encapsulated EVs (10 μg/ml) with or without the modification of stearyl-r8 (16 μM) (r8-SAP-EV and SAP-EV, respectively) for 72 h at 37 °C. The concentration of saporin encapsulated in 10 μg/ml EVs was estimated to be approximately 43.5 ng/ml using the FITC-labeled saporin. (c) Cell viability was analyzed using the WST-1 assay under the same experimental conditions as in (b). The data are expressed as the average (±SD) of four experiments. ^***p < 0.001.