Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module

Abstract

Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

Figure 1. Generation of engineered EXPLOR.

(a) Schematics of DNA constructs used for the production of EXPLOR. (b) Schematic showing fusion proteins and their proposed action. (c) HEK293T cells were transiently transfected with CIBN-EGFP-CD9 and mCherry-CRY2 expression vectors. The mCherry fluorescence was imaged before and after 488-nm laser stimulation (15 s in duration, 350 μW cm⁻²). Scale bars, 20 μm (5 μm for inset images). A representative result from at least 10 experiments. (d) HEK293T cells transiently transfected with CIBN-EGFP-CD9 and mCherry-CRY2 were imaged for time-lapse imaging of mCherry fluorescence for varying time periods (0–12 min) after a stimulation (black arrow) of 488-nm light (15 s in duration, 350 μW cm⁻²). Scale bars, 5 μm. A representative result of at least 10 experiments. (e) Quantification of mCherry fluorescence in the cytoplasm and at the plasma membrane. Data are presented as the mean±s.e.m. (n=3).

Figure 2. Light-dependent loading of target proteins in EXPLORs.

(a) Cells transiently transfected with CIBN-EGFP-CD9 and mCherry-CRY2 expression vectors were maintained under blue light illumination of varying powers for 48 h. Cell-derived exosomes were subject to immunoblot analysis using antibodies against mCherry, EGFP and CD63, an exosome marker. A representative result from three independent experiments. (b) The graph presents densitometry analysis for normalized amount of mCherry-CRY2 protein over CIBN-EGFP-CD9 protein from three independent experiments. Data are presented as the mean±s.e.m. (n=3), and Tukey's post hoc test was applied to significant group effects (**P<0.01, ***P<0.001) identified by analysis of variance.

Figure 3. Comparison of the exosome-loading capacity of target proteins between various protein-loading methods.

(a) HEK293T cells were transiently transfected with luciferase-mCherry-CRY2 expression vector alone, XPack-luciferase-mCherry expression vector or co-transfected with CIBN-EGFP-CD9 and luciferase-mCherry-CRY2 expression vectors. After 24 h, cells were imaged by fluorescence microscopy for the expression profile of mCherry fusion proteins. A representative result from five independent experiments. Scale bars, 20 μm. (b) Quantification of mCherry fluorescence. Data are presented as the mean±s.e.m. (n=5), and Tukey's post hoc test was applied to significant group effects identified by analysis of variance (ANOVA). Control: untransfected HEK293T cells; OVER: cells transiently transfected with a luciferase-mCherry-CRY2 vector; XP: cells transfected with an XPACK-luciferase-mCherry vector; and EXPLOR: cells transfected with both luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 vectors. (c) Cells transiently transfected with various vectors were maintained for 48 h. In the case of EXPLOR-producing cells, cells were maintained in the absence (OFF) or presence (ON) of blue light illumination; 5 × 10⁸ particles of the isolated exosomes were analysed for luciferase activity. Data are presented as the mean±s.e.m. (n=3), and Tukey's post hoc test was applied to significant group effects (**P<0.01, ***P<0.001) identified by ANOVA. (d) Loading efficiency of various protein-loaded exosomes was calculated by dividing the number of luciferase molecules in exosomes with the number of luciferase molecules in the exosome-producing cells. Numbers of luciferase molecules were estimated from a standard curve using recombinant luciferase. Data are presented as the mean±s.e.m. (n=3), and Tukey's post hoc test was applied to significant group effects (***P<0.001) identified by ANOVA. NS, not significant.

Figure 4. EXPLOR-mediated intracellular delivery of cargo proteins.

(a,b) HeLa cells were incubated in the absence or presence of 5 × 10⁹ particles of various isolated exosomes for 24 h and imaged by fluorescence microscopy. The fluorescence intensities of mCherry were quantified by two imaging processing tools, ImageJ and Cellprofiler. Data are presented as the mean±s.e.m. (n=15), and Tukey's post hoc test was applied to significant group effects (***P<0.001) identified by analysis of variance (ANOVA). Scale bars, 100 μm. (c,d) HeLa cells were incubated in the absence or presence of 0.1 mg ml⁻¹ mCherry:EXPLORs or Bax-mCherry:EXPLORs for 12 h, and fixed with 4% paraformaldehyde. Then, cytochrome c was stained with an antibody conjugated with Alexa Fluor 647 and imaged by confocal microscopy. The ratios of cytochrome c localization were analysed by cell counting. Data are presented as the mean±s.e.m. (n=3), and Tukey's post hoc test was applied to significant group effects (**P<0.01) identified by ANOVA. Scale bars, 20 μm. (e) HeLa cells were incubated in the absence or presence of 0.1 mg ml⁻¹ mCherry:EXPLORs or srIκB:EXPLORs for 12 h, treated with 10 ng ml⁻¹ tumour necrosis factor- α (TNF-α) for an additional 30 min, and fixed with 4% paraformaldehyde. NF-κB p65 was stained with an antibody conjugated with Alexa Fluor 488 and imaged by confocal microscopy. (f) The nuclear extracts of cells were assayed for the DNA-binding activity of p65/c-Rel (NF-κB). Data are presented as the mean±s.e.m. (n=3), and Tukey's post hoc test was applied to significant group effects (**P<0.01) identified by ANOVA. Scale bars, 20 μm. NS, not significant.

Figure 5. EXPLOR-mediated delivery of Cre recombinase in vitro and in vivo.

(a,b) Differentiated neurosphere-derived cells were incubated in the absence or presence of 2 × 10¹⁰ particles per ml of Cre:EXPLORs (0.16 mg ml⁻¹) or transfected with pCMV-Cre vector for 72 h. Cells were fixed with 4% paraformaldehyde and immune-stained with antibodies against a neuron-specific class III beta-tubulin marker, Tuj1, GFP and Hoechst 33342. The ratios of EGFP-expressing cells were analysed by cell counting. Data are presented as the mean±s.e.m. (n=10 fields), and Tukey's post hoc test was applied to significant group effects identified by ANOVA. Scale bars, 100 μm. (c) An experimental scheme for the administration of Cre:EXPLORs in loxp-stop-loxp-eNpHR3.0-EYFP transgenic mice. In total, 50 μl of Cre:EXPLORs (10 mg ml⁻¹) were administered to pCAG-loxP-STOP-loxP-eNpHR3.0-EYFP transgenic mice by ventrolateral injection. (d) Brain slices of EXPLOR-injected transgenic mice were fixed with 4% formaldehyde and imaged by fluorescence microscopy. Green fluorescence indicates eNpHR3.0-EYFP protein expression and blue fluorescence indicates cell nuclei. Inset images showed confocal microscopy images of the detailed cellular eNpHR3.0-EYFP expression in EXPLOR-administered mouse neurons of the zona incerta (ZI) region. Scale bars, 500 μm (50 μm for inset confocal images). Hip, hippocampus; Th, thalamus. A representative of two independent experiments. (e) Representative image of NeuN/GFAP immunohistochemistry of the brain. Pink, neuronal-specific nuclear protein (NeuN)-positive neurons; Red, glial fibrillary acidic protein (GFAP)-positive astrocyte cells. Objective lens, × 40. Scale bar, 20 μm. NS, not significant.

Figure 6. Schematic diagram of EXPLOR technology.

In EXPLOR-producing donor cells, CRY2 protein was fused to a cargo protein, and CIBN was conjugated with a representative marker of exosomes, CD9 protein. Blue light illumination induces the reversible PPI between CIBN and CRY2 fusion proteins. With continuous blue light irradiation, the cargo proteins are guided to the inner surface of the cell membrane or the surface of early endosomes. Mature multi-vesicular bodies (MVBs) then readily secrete cargo protein-carrying exosomes (EXPLORs) from the cells by membrane fusion with the plasma membrane. After exocytosis, EXPLORs can be easily isolated and purified in vitro. Purified EXPLORs can be used for delivery of the cargo proteins into target cells via membrane fusion or endocytosis processes. Bottom grey boxes highlight the essential steps from EXPLORs biogenesis to target cell delivery.