Systematic Comparisons of Formulations of Linear Oligolysine Peptides with siRNA and Plasmid DNA

Abstract

The effects of lysine peptide lengths on DNA and siRNA packaging and delivery were studied using four linear oligolysine peptides with 8 (K8), 16 (K16), 24 (K24) and 32 (K32) lysines. Oligolysine peptides with 16 lysines or longer were effective for stable monodisperse particle formation and optimal transfection efficiency with plasmid DNA (pDNA), but K8 formulations were less stable under anionic heparin challenge and consequently displayed poor transfection efficiency. However, here we show that the oligolysines were not able to package siRNA to form stable complexes, and consequently, siRNA transfection was unsuccessful. These results indicate that the physical structure and length of cationic peptides and their charge ratios are critical parameters for stable particle formation with pDNA and siRNA and that without packaging, delivery and transfection cannot be achieved.

Keywords: DNA delivery; RNA interference; biophysical characteristics; gene therapy; oligolysine peptide; siRNA delivery.

Figure 1

The binding properties of the linear lysine peptides with plasmid DNA or siRNA. The linear lysine peptides (K8, K16, K24 and K32) were mixed with 5 kb pDNA (pCI‐Luc) or siRNA at different N/P ratios for 30 min. The complexes were then run on the 1% agarose gel for the pDNA complexes and 4% agarose gel for the siRNA complexes subsequently. (A) The K8 pDNA and K16 pDNA complexes, (B) the K24 pDNA and K32 pDNA complexes, (C) the K8 siRNA and K16 siRNA complexes and (D) the K24 siRNA and K32 siRNA complexes. The formulations of the complexes are expressed in an N/P ratio.

Figure 2

The binding of the linear lysine peptides to (A) pDNA and (B) siRNA. The linear lysine peptides were mixed with PicoGreen‐labelled pDNA or siRNA at different N/P ratios for 30 min. The fluorescence intensity of the complexes was then measured and normalized with the naked pDNA or siRNA. The formulations of the complexes are expressed as an N/P ratio. * denotes a significant difference between the relative fluorescence unit (RFU) of the K8 pDNA/siRNA complexes and the rest of the complexes (p < 0.05).

Figure 3

The dissociation properties of linear lysine pDNA complexes. K8, K16, K24 and K32 were mixed with PicoGreen‐labelled pDNA at different N/P ratios for 30 min. Different concentrations of heparin were added to the complexes, and the fluorescence intensity of the complexes was measured. (A) The K8 pDNA complexes, (B) the K16 pDNA complexes, (C) the K24 pDNA complexes and (D) the K32 pDNA complexes. The formulations of the complexes are expressed as an N/P ratio.

Figure 4

The dissociation properties of linear lysine siRNA complexes. K8, K16, K24 and K32 were mixed with PicoGreen‐labelled siRNA at different N/P ratios for 30 min. Different concentrations of heparin were added to the complexes, and the fluorescence intensity of the complexes was measured. (A) The K8 siRNA complexes, (B) the K16 siRNA complexes, (C) the K24 siRNA complexes and (D) the K32 siRNA complexes. The formulations of the complexes are expressed as an N/P ratio.

Figure 5

The physical structure of the pDNA complexes. The complexes were prepared at an N/P ratio of 3:1 and were applied onto a glow‐discharged 300‐mesh copper grid coated with a Formvar/carbon support film. The complexes were stained with 1% uranyl acetate before blotting with filter paper and air‐dried. (A) The K8 pDNA complexes, (B) the K16 pDNA complexes, (C) the K24 pDNA complexes and (D) the K32 pDNA complexes. The bar represents 100 nm.

Figure 6

The physical structure of the pDNA complexes. The complexes were prepared at an N/P ratio of 4:1 and were applied onto a glow‐discharged 300‐mesh copper grid coated with a Formvar/carbon support film. The complexes were stained with 1% uranyl acetate before blotting with filter paper and air‐dried. (A) siRNA alone, (B) the K8 siRNA complexes, (C) the K16 siRNA complexes, (D) the K24 siRNA complexes and (E) the K32 siRNA complexes. The bar represents 100 nm.

Figure 7

Cellular binding/uptake efficiencies of linear lysine pDNA complexes. Neuro‐2A cells were seeded 24 h before transfection. The complexes were made by mixing the peptides with Cy5‐labelled pCI‐Luc for 30 min. Following the removal of full growth medium, complexes were overlaid to the cells for 4 h. The cells were harvested for analysis after transfection. For flow cytometry analysis, propidium iodide (PI) was used to estimate the cell viability following transfection: (A) cells exposed to K8 pDNA complexes at 3:1 N/P ratio, (B) cells exposed to K16 pDNA complexes at 3:1 N/P ratio, (C) cells exposed to K24 pDNA complexes at 3:1 N/P ratio and (D) cells exposed to K32 pDNA complexes at 3:1 N/P ratio.

Figure 8

Cellular uptake and localization of linear lysine pDNA complexes. Neuro‐2A cells were transfected at an N/P ratio of 3:1 as described in Figure 7. For confocal microscopy, cells were washed and stained with phalloidin for the F‐actin on the cell membrane (green), DAPI for the nucleus (blue) and Cy5 for the complexes (red). (A) Untreated cells, (B) cells exposed to K8 pDNA complexes, (C) cells exposed to K16 pDNA complexes, (D) cells exposed to K24 pDNA complexes and (E) cells exposed to K32 pDNA complexes. Scale bar = 15 μm.

Figure 9

Plasmid transfection efficiency mediated by linear lysine pDNA complexes. Neuro‐2A cells were seeded 24 h before transfection. The complexes were made by mixing peptides (K8, K16, K24 and K32) with pCI‐Luc in different N/P ratios for 30 min. Following removal of the full growth medium, complexes were overlaid to the cells for 4 h. After removing the transfection complexes, the full growth medium was added to the cells. Luciferase expression in the cells was analysed 24 h post‐transfection to estimate the transfection efficiencies of the complexes. The formulations of the complexes are expressed as an N/P ratio. * denotes the significant difference in the RLU/mg between the untreated cells and the transfected cells (p < 0.05).

Figure 10

Cytotoxicity induced by transfection of linear lysine pDNA complexes. Neuro‐2A cells were seeded 24 h before transfection. The complexes were made by mixing peptides (K8, K16, K24 and K32) with pCI‐Luc in different N/P ratios for 30 min. Following removal of the full growth medium, complexes were overlaid to the cells for 4 h. After removing the transfection complexes, the full growth medium was added to the cells. Cell viability was analysed 24 h post‐transfection to estimate the cytotoxicity caused by the transfection. The formulations of the complexes are expressed as an N/P ratio. * and *** denote the significant difference in the cell viability between the untreated cells and the transfected cells at p < 0.05 and p < 0.001, respectively.