Endosomal escape and siRNA delivery with cationic shell crosslinked knedel-like nanoparticles with tunable buffering capacities

Abstract

Cationic shell crosslinked knedel-like nanoparticles (cSCKs) have emerged as a highly efficient transfection agent for nucleic acids delivery. In this study, a new class of cSCKs with tunable buffering capacities has been developed by altering the amounts of histamines and primary amines incorporated into their crosslinked shell regions. The effect of histamine content of these nanoparticles with a hydrodynamic diameter of ca. 20 nm, on the siRNA-binding affinity, cytotoxicity, immunogenicity, and transfection efficiency was investigated. The modification of cSCKs with histamine was found to reduce the siRNA-binding affinity and cellular binding. On the other hand, it significantly reduced the toxicity and immunogenicity of the nanoparticles with subsequent increase in the transfection efficiency. In addition, escape from endosomes was facilitated by having two species of low and high pK(a)s (i.e. histamine and primary amine groups, respectively), as demonstrated by the potentiometric titration experiments and the effect of bafilomycin A1, an inhibitor of the endosomal acidification, on the transfection efficiency of cSCKs. Histamine modification of 15 mol% was a threshold, above which cSCKs with higher histamine content completely lost the ability to bind siRNA and to transfect cells. This study highlights the potential of histamine incorporation to augment the gene silencing activity of cationic nanoparticles, reduce their toxicity, and increase their biocompatibility, which is of particular importance in the design of nucleic acids delivery vectors.

Fig. 1

DLS histograms from measurements of nanoparticles with various amounts of primary amines and histamines: (A) 0%-His-cSCK (12), (B) 15%-His-cSCK (13), (C) 50%-His-cSCK (14) and (D) 100%-His-micelles (11).

Fig. 2

(A) Zeta-potential measurements of micelles 8–11 with increasing histamine content, each at pH 5.0, 6.0 and 7.4. (B) Relative buffering capacity of cSCK nanoparticles 12–14 and micelles 11 containing various amounts of histamine obtained from potentiometric titration experiments, and comparison with PEI. Each bar represents the amount of HCl (in mL) needed to change the pH of the nanoparticle solutions from pH 7.4 to 5.1.

Fig. 3

The expression of IL-3, IL-6, IL-9, IL-10, IL-12(p40), IL-13, Eotaxin, RANTES, MCP-1, MIP-1β, KC and TNF-α were particularly enhanced upon the treatment of Raw 264.7 mouse macrophages with the unmodified-cSCKs (0%-His-cSCKs) for 24 h, as compared to the 15%-His-cSCKs and the control-untreated cells.

Fig. 4

Gel-shift assay of Cy3-labeled siRNA, either naked (N/P = 0) or complexed to micelles or cSCKs of various composition at increasing N/P ratios from 0.25–6. Complete binding is observed at N/P ratios of 2 and 4, for 0% and 15% histamine-modified cSCKs, whereas no binding was observed for cSCKs and micelles that contain 50% and 100% histaminemodified shells, respectively.

Fig. 5

Laser scanning confocal microscopy analysis of the control-untreated RAW 264.7 mouse macrophages (A), cells treated with Cy3-siRNA (100 nm) complexed with Lipofectamine (B), 0%-His- (C), 15%-His- (D), 50%-His- (E) and 100%-His-nanoparticles (F) at N/P ratio of 5. Two (left panel) and three (right panel)-dimensional images were collected for the cells (A–D), while only two-dimensional images were collected for E and F. On the two-dimensional images, the nucleus were stained with DRAQ5 nuclear stain (blue panel), whereas Cy3-labeled siRNA appears in red. The transmitted light-images and merged images are also indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Transfection efficiency of death-siRNA complexed with Lipofectamine or cSCKs of varying primary amine and histamine ratios into OVCAR-3 cells and RAW 264.7 mouse macrophages at N/P ratios of 5 and 10. (A) The transfection of the death-siRNA/cSCK complexes in the two different cell lines at an N/P ratio of 5. (B) The effect of N/P ratio on the transfection efficiency in the OVCAR-3 cells, determined by comparison of death-siRNA/cSCKs vs. negative control-siRNA/cSCKs. (C) The effect of N/P ratio on the cytotoxicity of cSCKs in OVCAR-3 cells, determine by comparison of negative control-siRNA/cSCKs vs. control, untreated cell assays.

Fig. 7

Transfection efficiency of death-siRNA (100 nm) complexed with Lipofectamine or cSCKs of varying compositions into OVCAR-3 cells, with and without pre-treatment with bafilomycin A1 (BA, 200 nm for 30 min before the transfection and continued during the transfection).

Scheme 1

Incorporation of various amounts of primary amines and histamines onto a segment of an amphiphilic block copolymer backbone by a sequential two-step process of amidation followed by deprotection.

Scheme 2

Self assembly of polymers with various amounts of primary amines and histamines (4–7) into micelles (8–11) followed by crosslinking selectively in the hydrophilic shell regions of (8–10) to obtain cSCKs (12–14).