Intracellular delivery of proteins by nanocarriers

Abstract

Intracellular delivery of proteins is potentially a game-changing approach for therapeutics. However, for most applications, the protein needs to access the cytosol to be effective. A wide variety of strategies have been developed for protein delivery, however access of delivered protein to the cytosol without acute cytotoxicity remains a critical issue. In this review we discuss recent trends in protein delivery using nanocarriers, focusing on the ability of these strategies to deliver protein into the cytosol.

Keywords: cytosolic delivery; nanoparticle-stabilized nanocapsules; nuclear targeting; protein; punctate fluorescence.

Figure 1.. Intracellular delivery of TALE and Cas9 proteins using cationic lipids.

(A) Recombinases, TALE proteins and Cas9 endonucleases fuse with supernegatively charged proteins, (-30) GFP, first, and complex with cationic lipids, resulting highly anionic proteins or protein–nucleic acid complexes and mediating their delivery into mammalian cells. (B) T7EI assay of simultaneous genome modification at eGFP and three endogenous genes in U2OS cells 48 h after a single treatment of 100 nM Cas9 protein, 25 nM of each of the four sgRNAs shown (100 nM total sgRNA) and 0.8 ml RNAiMAX. (C) Cas9-mediated gene disruption results in the loss of GFP expression when visualized 10 days later. The upper panels show GFP signal only, whereas lower panels include additional immune-histological markers. Yellow boxes in the lower panels highlight hair cells that have lost GFP expression. All scale bars (white), 10 μm. GFP: Green fluorescent protein. Adapted with permission from [27].

Figure 2.

(A) Calculated electrostatic surface potentials of +36 GFP, colored from -25 kT/e (dark red) to +25 kT/e (dark blue). (B) Confocal fluorescence microscopy of live cells incubated with 100 nM +36 GFP–mCherry for 4 h at 37°C. Red color represents mCherry signal; green color represents +36 GFP signal. The scale bar is 15 μm. (C) Fluorescence microscopy of a retinal section of a CD1 adult mouse injected with 0.5 μl of 100 μM +36 GFP. The retina was harvested and analyzed 6 h after injection. GFP fluorescence is shown in green and DAPI nuclear stain is shown in blue. Lower right: retinal sections of neonatal RC::PFwe mouse pups harboring a nuclear LacZ reporter of Cre activity. Three days after injection of 0.5 μl of 40 μM +36 GFP–Cre, retinas were harvested, fixed and stained with X-gal. Dots on the graph represent the total number of recombined cells counted in each retina. DAPI: 4′,6-Diamidino-2-phenylindole; GFP: Green fluorescent protein. Adapted with permission from [36,37].

Figure 3.. Preparation and delivery of nanogel-caspase-3 conjugates.

(A) Preparation of nanogel–caspase-3 conjugates: covalent conjugation of caspase-3 in the interior or on the surface of polymeric redox-sensitive nanogels through disulfide linkages. (B) Cellular internalization: NG-FITC–Casp–InRRR and NG-FITC–Casp–OutRRR at 0.5 mg/ml on HeLa cells. The images are overlap of FITC channel (green; caspase-3), DRAQ5 channel (red; nucleus) and differential interference contrast. This experiment was performed with triplicate visualization on 1 day. One representative field is shown for each condition. FITC: Fluorescein isothiocyanate. Adapted with permission from [45].

Figure 4.. Delivery of large anionic protein β-gal (473 kDa) with gold nanoparticles into cells.

(A) Intracellular delivery of functional protein using gold nanoparticles. (B) Colocalization study using confocal microscopy after protein transfection (NP_Pep/FITC-gal: 100 nM/50 nM) of HeLa cells in the presence of FM4-64: (a) green fluorescence from FITC-gal, (b) red fluorescence from FM4-64, an endosome-specific marker and (c) overlap of the green and the red channels. In the merged image, green spots (shown with green arrowheads) indicate proteins outside endosomes, while entrapped proteins inside endosomes appear as yellow dots (shown with yellow arrowheads). Adapted with permission from [59].

Figure 5.. Intracellular protein delivery by nanoparticle-stabilized nanocapsules.

(A) Schematic showing the preparation of the protein NPSC complex containing caspase-3 or GFP and proposed delivery mechanism. (B) Live cell imaging of rapid GFP release into the cytosol of HeLa cell by NPSCs. Scale bar: 20 μm. AuNP: Gold nanoparticle; GFP: Green fluorescent protein; NPSC: Nanoparticle-stabilized nanocapsule. Adapted with permission from [60].

Figure 6.. Delivery of eGFP fused with nuclear localization signals to cells using nanoparticle-stabilized nanocapsules.

(A) Schematic representing the cytosolic delivery and nuclear accumulation of proteins with NLSs. (B) Structure of enhanced (e)GFP fused with NLSs. (C) LSCM images showing different cellular distribution patterns of eGFP fused with NLSs. Bars: 20 μm. (D) Time-lapse LSCM images unveil the kinetics of nuclear import of NLS^c-Myc–eGFP. GFP: Green fluorescent protein; LSCM: Laser scanning confocal microscope; NLS: Nuclear localization signal. Adapted with permission from [62].

Figure 7.. Schematic representation of intracellular delivery of proteins via nanocarriers.