Extracellular Microvesicles Modified with Arginine-Rich Peptides for Active Macropinocytosis Induction and Delivery of Therapeutic Molecules

Abstract

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), transfer bioactive molecules from donor to recipient cells in various pathophysiological settings, thereby mediating intercellular communication. Despite their significant roles in extracellular signaling, the cellular uptake mechanisms of different EV subpopulations remain unknown. In particular, plasma membrane-derived MVs are larger vesicles (100 nm to 1 μm in diameter) and may serve as efficient molecular delivery systems due to their large capacity; however, because of size limitations, receptor-mediated endocytosis is considered an inefficient means for cellular MV uptake. This study demonstrated that macropinocytosis (lamellipodia formation and plasma membrane ruffling, causing the engulfment of large fluid volumes outside cells) can enhance cellular MV uptake. We developed experimental techniques to induce macropinocytosis-mediated MV uptake by modifying MV membranes with arginine-rich cell-penetrating peptides for the intracellular delivery of therapeutic molecules.

Keywords: arginine-rich cell-penetrating peptides; endocytosis; exosomes; extracellular vesicles; macropinocytosis; microvesicles; peptide modification.

Figure 1

Induction of macropinocytosis by EGFR activation enhances cellular MV uptake. (A) Schematic representation of macropinocytosis induction with lamellipodia formation and plasma membrane ruffling by EGFR activation, which enhances MV uptake. (B) Measurement of particle size of fluorescence-labeled MVs (FL-MVs). (C, D) Relative A431 cellular uptake of the macropinocytosis marker FITC-dextran (molecular weight: 70 000) (0.5 mg/mL) (C) or FL-MV (125 particles/μL) (D) cotreatment with or without EGF (100 nM) in 10% FBS-containing cell culture medium for 24 h at 37 °C prior to flow cytometric analysis. The flow cytometry data have been expressed as the means (±SD) of three independent experiments. ***P < 0.001, ****P < 0.0001. (E) Confocal laser microscopy observation of A431 cells treated with FL-MVs (125 particles/μL) with or without EGF cotreatment (100 nM) in a 10% FBS-containing cell culture medium for 24 h at 37 °C. Green signals: FL-MVs; blue signals: Hoechst 33 342. Scale bar: 50 μm (EGF: epidermal growth factor; SD: standard deviation; FBS: fetal bovine serum).

Figure 2

Modification of arginine-rich CPPs significantly enhances the cellular uptake of MVs via macropinocytosis. (A) Schematic representation: cellular uptake of MVs is enhanced via macropinocytosis induction following stearyl–octaarginine (stearyl–R8) modification. (B) Transmission electron microscopy (TEM) observation of MVs with stearyl–R8 modification. Scale bar: 1000 nm. (C) Particle size measurement of FL-MVs with stearyl–R8 modification. (D) Observation of A431 cells treated with FL-MVs (240 particles/μL) with or without stearyl–R8 modification (20 μM) in a 10% FBS-containing cell culture medium for 24 h at 37 °C using a confocal laser microscope. Green signals: FL-MVs, blue signals: Hoechst 33 342. Scale bar: 50 μm. (E) Relative A431 cellular uptake of FL-MVs (240 particles/μL) with or without stearyl–R8 modification (20 μM) in a 10% FBS-containing cell culture medium for 24 h at 37 °C before flow cytometry analysis. The flow cytometry data are expressed as means (±SD) of three experiments. **P < 0.01.

Figure 3

Induction of actin reorganization, lamellipodia formation, and macropinocytosis by stearyl–R8-modified MVs. (A) Relative A431 cellular uptake of the macropinocytosis marker, FITC-dextran (molecular weight: 70 000) (0.5 mg/mL), with or without MV cotreatment (240 particles/μL) with stearyl–R8 modification (10 μM) in a 10% FBS-containing cell culture medium for 24 h at 37 °C before flow cytometry. (B) Relative A431 cellular uptake of stearyl–R8 (10 μM)-modified FL-MVs (240 particles/μL) in the presence or absence of the macropinocytosis inhibitor EIPA (100 μM) in a 10% FBS-containing cell culture medium for 1 h at 37 °C before flow cytometry. The flow cytometry data have been expressed as means (±SD) of three experiments. ***P < 0.001. (C) Cellular staining (A431 cells) with rhodamine-phalloidin and observation after cellular treatment of MVs (240 particles/μL) with or without stearyl–R8 modification (10 μM) in a 10% FBS-containing cell culture medium for 1 h at 37 °C using a confocal laser microscope. Red signals: rhodamine-phalloidin. Scale bar: 50 μm. Arrows indicate representative lamellipodia formations.

Figure 4

Dependence of proteoglycans on stearyl–R8-modified MV uptake. (A, B) Relative cellular uptake (CHO-K1 cells: wild-type, CHO-A745: all glycosaminoglycan-deficient) of FL-MVs (240 particles/μL) with (B) or without (A) stearyl–R8 modification (10 μM) in a 10% FBS-containing cell culture medium for 24 h at 37 °C prior to flow cytometry. The flow cytometry data are expressed as means (±SD) of three independent experiments. ****P < 0.0001. (C) Cellular staining (A431 cells) with anti-syndecan-4 antibody and confocal laser microscopic observation following MV cellular treatment (240 particles/μL) with or without stearyl–R8 modification (10 μM) in a 10% FBS-containing cell culture medium for 1 h at 37 °C. Arrows show representative cluster formations of syndecan-4. Green signals: anti-syndecan-4 antibody. Scale bar: 20 μm.

Figure 5

Enhanced anticancer activity of curcumin derivatives complexed with stearyl–R8-modified MVs. (A) Chemical structure of the curcumin derivative GO-Y078. (B) Viability of A431 cells treated with GO-Y078 (10 μM) complexed with or without stearyl–R8 modified (10 μM) MVs (480 particles/μL) in a 10% FBS-containing cell culture medium for 72 h at 37 °C prior to WST-8 assay. The WST-8 data are expressed as means (±SD) of four experiments. **P < 0.01, ***P < 0.001. (C) Microscopic observation (enlarged) of A431 cells with or without GO-Y078 (10 μM) complexed with stearyl–R8 modification (10 μM) in MVs (480 particles/μL) in a 10% FBS-containing cell culture medium for 72 h at 37 °C. Supporting Figure S6 depicts the original images.