Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins

Abstract

Nucleic acid reagents, including small interfering RNA (siRNA) and plasmid DNA, are important tools for the study of mammalian cells and are promising starting points for the development of new therapeutic agents. Realizing their full potential, however, requires nucleic acid delivery reagents that are simple to prepare, effective across many mammalian cell lines, and nontoxic. We recently described the extensive surface mutagenesis of proteins in a manner that dramatically increases their net charge. Here, we report that superpositively charged green fluorescent proteins, including a variant with a theoretical net charge of +36 (+36 GFP), can penetrate a variety of mammalian cell lines. Internalization of +36 GFP depends on nonspecific electrostatic interactions with sulfated proteoglycans present on the surface of most mammalian cells. When +36 GFP is mixed with siRNA, protein-siRNA complexes approximately 1.7 mum in diameter are formed. Addition of these complexes to five mammalian cell lines, including four that are resistant to cationic lipid-mediated siRNA transfection, results in potent siRNA delivery. In four of these five cell lines, siRNA transfected by +36 GFP suppresses target gene expression. We show that +36 GFP is resistant to proteolysis, is stable in the presence of serum, and extends the serum half-life of siRNA and plasmid DNA with which it is complexed. A variant of +36 GFP can mediate DNA transfection, enabling plasmid-based gene expression. These findings indicate that superpositively charged proteins can overcome some of the key limitations of currently used transfection agents.

Fig. 1.

Supercharged GFP variants and their ability to penetrate cells. (A) Calculated electrostatic surface potentials of GFP variants used in this work, colored from −25 kT/e (dark red) to +25 kT/e (dark blue). (B) Flow cytometry analysis showing amounts of internalized GFP in HeLa cells treated with 200 nM each superpositive GFP variant and washed three times with PBS containing heparin to remove cell surface-bound GFP. (C) Flow cytometry analysis showing amounts of internalized +36 GFP (green) in HeLa, IMCD, 3T3-L, PC12, and Jurkat cells compared with untreated cells (black).

Fig. 2.

Mechanistic probes of +36 GFP internalization. (A) Internalization of +36 GFP in HeLa cells after coincubation for 1 h at 37 °C. (B) Inhibition of +36 GFP cell penetration in HeLa cells incubated at 4 °C for 1 h. Cells were only partially washed to enable +36 GFP to remain partially bound to the cell surface. (C and D) +36 GFP internalization under the conditions in A but in the presence of caveolin-dependent endocytosis inhibitors filipin and nystatin, respectively. (E) +36 GFP internalization under the conditions in A but in the presence of the clathrin-dependent endocytosis inhibitor chlorpromazine. (F) Localization of Alexa Fluor 647-labeled transferrin (red) and +36 GFP (green) 20 min after endocytosis. (G) Inhibition of +36 GFP internalization in HeLa cells in the presence of the actin polymerization inhibitor cytochalasin D. (H) Inhibition of +36 GFP internalization in HeLa cells treated with 80 mM sodium chlorate. (I) Internalization of +36 GFP in CHO cells incubated at 37 °C for 1 h. (J) Lack of +36 GFP internalization in PDG-CHO cells. (I and J) Cell nuclei were stained with DAPI (blue). [Scale bars, 10 μm (A–H) and 20 μm (I–J).]

Fig. 3.

Binding and transfection efficiency of siRNA by supercharged GFPs. (A) Gel-shift assay (31) to determine superpositive GFP:siRNA binding stoichiometry. Ten picomoles of siRNA was mixed with various molar ratios of each GFP for 10 min at 25 °C and then analyzed by nondenaturing PAGE. GFP–siRNA complexes were observed to remain in the loading well of the gel. (B) Flow cytometry analysis showing levels of siRNA in HeLa cells treated with a mixture of 50 nM Cy3-siRNA and 200 nM +15, +25, or +36 GFP, followed by three heparin washes to remove noninternalized GFP (Fig. S1 in the SI Appendix). (C) Flow cytometry analysis showing levels of Cy3-labeled siRNA delivered into HeLa, IMCD, 3T3-L, PC12, and Jurkat cells after incubation with a mixture of 50 nM Cy3-siRNA and either ≈2 μM Lipofectamine 2000 (blue) or 200 nM +36 GFP (green) compared with cells treated with siRNA without transfection reagent (black). (D) Fluorescence microscopy images of stably adherent cell lines (HeLa, IMCD, and 3T3-L) 24 h after a 4-h treatment with 200 nM +36 GFP and 50 nM Cy3-siRNA. Each image is an overlay of three channels: blue (DAPI stain), red (Cy3-siRNA), and green (+36 GFP); yellow indicates the colocalization of red and green. (Scale bar, 10 μm.)

Fig. 4.

Suppression of GAPDH mRNA and protein levels resulting from siRNA delivery. (A) GAPDH mRNA level suppression in HeLa cells 48, 72, or 96 h after treatment with 50 nM siRNA and ≈2 μM Lipofectamine 2000, or with 50 nM siRNA and 200 nM +36 GFP, as measured by RT-quantitative PCR. Suppression levels shown are normalized to β-actin mRNA levels; 0% suppression is defined as the mRNA level in cells treated with negative control siRNA. (B) GAPDH or β-actin protein level suppression in HeLa cells 48, 72, or 96 h after treatment with siRNA and ≈2 μM Lipofectamine 2000, or with siRNA and 200 nM +36 GFP. (C) GAPDH protein level suppression in HeLa, IMCD, 3T3-L, PC12, and Jurkat cells 96 h after treatment with 50 nM siRNA and ≈2 μM Lipofectamine 2000, 200 nM +36 GFP, or 200 nM +36 GFP-HA2. Protein levels were measured by Western blotting and are normalized to β-tubulin protein levels; 0% suppression is defined as the protein level in cells treated with negative control siRNA. Values and error bars represent the mean ± SD of three independent experiments in A and five independent experiments in B and C. Independently prepared protein batches were tested (*, 5 batches; **, 3 batches).

Fig. 5.

The siRNA transfection activities of a variety of cationic synthetic peptides compared with that of +15 and +36 GFP. Flow cytometry was used to measure the levels of internalized Cy3-siRNA in HeLa cells treated for 4 h with a mixture of 50 nM Cy3-siRNA and the peptide or protein shown.

Fig. 6.

Plasmid DNA transfection into HeLa, IMCD, 3T3-L, PC12, and Jurkat cells by Lipofectamine 2000, +36 GFP, or +36 GFP-HA2. Cells were treated with 800 ng of pSV-β-galactosidase plasmid and 200 nM or 2 μM +36 GFP or +36 GFP-HA2 for 4 h. After 24 h, β-galactosidase activity was measured. Values and error bars represent the mean ± SD of three independent experiments.