Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9

International audiencePeptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine-and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization

Interaction of the conjugates with cell surface glycosaminoglycans.

<p>CHO K1 and CHO 745 cells were treated with 1 µM of the indicated conjugates for 4 h. The fluorescence was quantified by FACS. The values are the means ± SD of at least three independent experiments.</p

Inhibition of luciferase expression by (R/W)9-ASPNA.

<p>Comparison of disulfide or maleimide linker containing (R/W)9-ASPNA in dose response. Relative luciferase activity in PPT/HeLa cells incubated with conjugated ASPNAs with (grey bars) or without (white bars) 150 µM chloroquine (CQ) for 4 h in serum-free Opti-MEM medium. Luciferase activity was measured 24 h after addition of doxycycline and is expressed as luminescence/mg of protein. Percentages are relative to luciferase activity in untreated cells. Values are the mean ± SD of at least three independent experiments. ASPNA, antisense PNA; SCRPNA, scramble PNA; S-S, disulfide linker; mal, maleimide linker.</p

Endocytosis pathways involved in (R/W)9-ASPNA-TR cell uptake.

<p>PPT/HeLa cells were incubated with Opti-MEM medium containing 1.5 µM (R/W)9-ASPNA-TR and (A) 25 µg/ml Alexa Fluor 488-transferrin, (B) 0.15 µM Alexa Fluor 488-dextran or (C) 10 µg/ml Alexa Fluor 488-cholera toxin subunit B at 37°C for 2 h and then analyzed. (D) Cells were incubated with 1.5 µM (R/W)9-ASPNA-TR conjugate for 4 h and grown for 18 h in conjugate-free medium. Lysotracker Green DND-26 was then added for 2 h and cells were observed using a Zeiss Axio Observer.Z1 microscope. Left panels show differential interference contrast images, middle panels fluorescence images, and right panels show overlays of the fluorescent images (i.e., merged green and red channels). Scale bar: 20 µm. Extents of co-localisation of (R/W)9-ASPNA-TR conjugate with the following were determined using the Icy imaging software (<a href="http://icy.bioimageanalysis.org/" target="_blank">http://icy.bioimageanalysis.org/</a>): Alexa Fluor 488-transferrin, 45.22±6.92%; dextran, 42±0.29%; cholera toxin, 47.91±0.01%; and Lysotracker Green DND-26, 48.85±5.09%.</p

Inhibition of luciferase expression by ASPNA conjugated to peptides.

<p>(A) Relative luciferase activity in PPT/HeLa cells incubated with conjugated or unconjugated ASPNA as indicated at 1.5 µM concentrations with (grey bars) or without (white bars) 150 µM chloroquine (CQ) for 4 h in serum-free Opti-MEM medium. Luciferase activity was measured 24 h after addition of doxycycline and expressed as luminescence/mg of protein. Percentages are relative to luciferase activity in untreated cells. Values are the mean ± SD of at least three independent experiments.</p

Effects of the chloroquine on (R/W)9-ASPNA internalization.

<p>(A) PPT/HeLa cells were incubated with 1 µM (R/W)9-ASPNA-TR with (+CQ) or without chloroquine (–CQ) for 4 h prior to microscopic observation. Images are the projection of all z-stacked signals (0.2 µm between each image). Light field (upper panels) and fluorescent images (lower panels) are shown. Scale bar: 30 µm. (B) Representative FACS data of cell uptake (TR fluorescence) of ASPNA-TR in the presence of CQ (red curve) or in the absence of CQ (black curve) and of (R/W)9-ASPNA-TR in the presence of CQ (yellow curve) or in the absence of CQ (blue curve). Fluorescence quantification (mean ± SD; n = 5) is shown in the histogram.</p

Kinetics and energy-dependence of (R/W)9-ASPNA-TR uptake into PPT/HeLa cells.

<p>(A) Cells were incubated with 1 µM (R/W)9-ASPNA-TR or with 1 µM ASPNA-TR for the indicated times. Fluorescence was measured by FACS, and data shown are the mean ± SD of three independent experiments. (B) PPT/HeLa cells were incubated with 1 µM (R/W)9-ASPNA-TR at 37°C or at 4°C or with 1 µM (R/W)9-ASPNA-TR and 10 mM sodium azide (NaN₃) and 6 mM deoxyglucose (DOG) to deplete cellular ATP at 37°C for 60 min. The histogram shows the fluorescence values (means ± SD of four independent experiments) measured by FACS analysis.</p

Effect on the substitution of (R/W)9 arginines by lysines on the uptake and antisense activity of the conjugated ASPNA.

<p>(A) PPT/HeLa cells were incubated with 1 µM (R/W)9-ASPNA-TR or 1 µM (K/W)9-ASPNA-TR conjugates for 4 h. Intracellular fluorescence was measured by FACS, and the background fluorescence of the cells incubated with ASPNA-TR was subtracted. Histograms show the quantitative determinations (means ± SD; n = 6). (B) PPT/HeLa cells were incubated with the indicated concentrations of (R/W)9-ASPNA-TR or (K/W)9-ASPNA-TR in serum-free Opti-MEM medium and 150 µM chloroquine for 4 h. Luciferase expression was induced by addition of 150 µL doxycycline. After 24 h cells were lysed and luciferase activity was measured. Values (means ± SD of at least seven independent experiments) were normalized to the average value of cells not treated with PNA.</p