Promises and pitfalls of intracellular delivery of proteins

Abstract

The direct delivery of functional proteins into the cell cytosol is a key issue for protein therapy, with many current strategies resulting in endosomal entrapment. Protein delivery to the cytosol is challenging due to the high molecular weight and the polarity of therapeutic proteins. Here we review strategies for the delivery of proteins into cells, including cell-penetrating peptides, virus-like particles, supercharged proteins, nanocarriers, polymers, and nanoparticle-stabilized nanocapsules. The advantages and disadvantages of these approaches including cytosolar delivery are compared and contrasted, with promising pathways forward identified.

Figure 1

Schematic illustration of protein delivery systems. (A) cell-penetrating peptides, (B) supercharged proteins, (C) virus-like particle, (D) nanocarrier, (E) liposomes, (F) polymer, and (G) nanoparticle-stabilized nanocapsule.

Figure 2

Protein delivery into cells by using a CPP (R7). (A) Schematic diagrams of recombinant proteins with or without the CPP (R7)-conjugated vectors. (B) Comparison of the efficiency of two different protein-delivery systems (CPP- and Streptolysin O-mediated). Transduction of GFP and R7-GFP was detected by confocal microscopy. GFP or R7-GFP is visualized in green. Nuclei were counter-stained with DAPI and the images were merged (the top three rows show 400× magnification and the bottom two rows show 1000× magnification plus 3× zoom). Scale bars represent 20 μm. GFP, green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole. Reprinted with permission from ref (11). Copyright 2011 Nature Publishing Group.

Figure 3

MNPs deliver serratiopeptidase into cells. (A) Scheme of the immobilization of Cytc-Lac into MSN-SH via redox-sensitive smart bonds followed by its intracellular delivery into cancer cells. (B) Internalization of the MSN-SPDP-Cyt c-Lac bioconjugate by HeLa cells observed by confocal microscopy. The left image is the autofluorescence image of the cells, the lower left shows the FITC labeled MSN internalized by the cells, the lower right shows the FM4-64 labeled endosomes, and the upper right micrograph is the merged image. FITC, fluorescein isothiocyanate. Reprinted with permission from ref (36). Copyright 2014 American Chemical Society.

Figure 4

PEI-based polyelectrolytes deliver protein into MCF7 cells. (A) Confocal images for intracellular tracking of the cationic polyelectrolyte/BSA complexes (WR1) and BSA (10 μg/mL) and (B) the intracellular endolysosome amount of the BSA- and polycation/BSA-treated cells. Free BSA was labeled with RITC (red). Acidic compartments and nuclei were stained with LysoTracker Green (green) and Hoechst 33342 (blue), respectively. RITC, Rhodamine Bisothiocyanate. Reprinted with permission from ref (51). Copyright 2013 American Chemical Society.

Figure 5

Nanoplex delivery of a large anionic protein β-galactosidase (473 kDa) into cells. (A) Intracellular delivery of functional protein using gold nanoparticles. (B) Co-localization 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). Reprinted with permission from ref (57). Copyright 2010 American Chemical Society.

Figure 6

Intracellular protein delivery by NPSCs. (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. Reprinted with permission from ref (62). Copyright 2013 American Chemical Society.