Brain Gene Silencing with Cationic Amino-Capped Poly(ethylene glycol) Polyplexes

Abstract

Therapeutic gene silencing in the brain is usually achieved using highly invasive intracranial administration methods and/or comparatively toxic vectors. In this work, we use a relatively biocompatible vector: poly(ethylene glycol) star-shaped polymer capped with amine groups (4APPA) via the nose to brain route. 4APPA complexes anti- itchy E3 ubiquitin protein ligase (anti-ITCH) siRNA to form positively charged (zeta potential +15 ± 5 mV) 150 nm nanoparticles. The siRNA-4APPA polyplexes demonstrated low cellular toxicity (IC50 = 13.92 ± 6 mg mL^-1) in the A431 cell line and were three orders of magnitude less toxic than Lipofectamine 2000 (IC50 = 0.033 ± 0.04 mg mL^-1) in this cell line. Cell association and uptake of fluorescently labelled siRNA bound to siRNA-4APPA nanoparticles was demonstrated using fluorescent activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM), respectively. Gene silencing of the ITCH gene was observed in vitro in the A431 cell line (65% down regulation when compared to the use of anti-ITCH siRNA alone). On intranasal dosing with fluorescently labelled siRNA-4APPA polyplexes, fluorescence was seen in the cells of the olfactory bulb, cerebral cortex and mid-brain regions. Finally, down regulation of ITCH was seen in the brain cells (54 ± 13% ITCH remaining compared to untreated controls) in a healthy rat model, following intranasal dosing of siRNA-4APPA nanoparticles (0.15 mg kg^-1 siRNA twice daily for 3 days). Gene silencing in the brain may be achieved by intranasal administration of siRNA- poly(ethylene glycol) based polyplexes.

Keywords: brain; gene silencing; intranasal; nanoparticles; polyethylene glycol (PEG); siRNA delivery.

Figure 1

Synthesis of 4APPA.

Figure 2

4APPA-siRNA polyplexes containing 20 μg mL⁻¹ siRNA in phosphate buffer (2 mM, pH = 6.0) (a) polyplex z-average mean size, (b) polyplex zeta potential, (c) polyplex polydispersity, all particle characterisation data are shown as mean ± s.d. (n = 3 separate experiments, * = statistically significant difference p < 0.05, ** = statistically significant difference p < 0.01, *** = statistically significant difference p < 0.001, **** = statistically significant difference p < 0.0001), (d) fluorescent activated cell sorting histograms from the FAM channel following incubation of FAM™-siRNA with A431 cells for 6 h (black = cells alone, yellow = cells plus FAM™-siRNA alone, pink = cells plus FAM™-siRNa, Lipofectamine, blue = 4APPA-FAM™-siRNA, (e) SEM image of 4APPA-siRNA at an N/P ratio of 43, (f) SEM image of 4APPA-siRNA at an N/P ratio of 32.

Figure 3

CLSM images (a,b,d–f) and in vitro gene silencing using siRNA (c) in A431 cells. (a,b,d–f)—A431 cells (seeded at 150,000 cells per well) with the cell nucleus stained blue (DAPI), cell cytoplasm stained red (CellMask™) and FAM™-siRNA stained green: (a) cells were treated with 4APPA-FAM™-siRNA (20 μg mL⁻¹ siRNA, 500 μL) for 6 h, (b) cells were treated with Lipofectamine-FAM™-siRNA (100 μg mL⁻¹ siRNA, 30 μL) according to the manufacturer’s instructions, (d) cells were treated with PBS, (e) cells were treated with FAM™-siRNA alone (20 μg mL⁻¹ siRNA, 500 μL), (f) untreated control cells. Bar = 20 μm. Data showing separate images from the FAM, DAPI and CellMask channels may be found in Figures S3–S7 in the Supplementary Information file. (c) Western blot densitometry analysis of ITCH gene silencing showing percentage ITCH down regulation when compared to siRNA alone (mean ± s.d., n = 5 and representative of 3 separate experiments) in A431 cells (seeded at 400,000 cells per flask) incubated for 6 h with 4APPA-siRNA (20 μg mL⁻¹ siRNA, 500 μL, N, P ratio = 32 in phosphate buffer—2 mM, pH = 6.0), Lipofectamine 2000™-siRNA (100 μg mL⁻¹ siRNA, 30 μL) prepared according to the manufacturer’s instructions, siRNA alone (20 μg mL⁻¹ siRNA, 500 μL), Western blot gels may be found in Figure S8 in the Supplementary Information file. * = statistically significant difference p < 0.05.

Figure 4

CLSM images (a–e) with the nucleus stained blue with DAPI and Cy³-siRNA-GAPDH stained red–arrowed and in vivo gene silencing (f) in rat brain tissue. Bar = 20 μm; All formulations were dosed in phosphate buffer (2 mM, pH = 6.0): (a) rat olfactory bulb 5 min after the intranasal delivery of Cy³-siRNA-GAPDH alone (1 mg mL⁻¹, 20 μL), (b) rat cerebral cortex 1 h after the intranasal administration of Cy³-siRNA-GAPDH alone (1 mg mL⁻¹, 20 μL), (c) rat mid brain 1 h after the intranasal administration of Cy³-siRNA-GAPDH alone (1 mg mL⁻¹, 20 μL), (d) rat olfactory bulb 5 min after the intranasal administration of 4APPA-Cy³-siRNA-GAPDH polyplexes (0.444 mg mL⁻¹ siRNA, 45 μL, N, P ratio = 32), (e) rat cerebral cortex 1 h after the intranasal administration of 4APPA-Cy³-siRNA-GAPDH polyplexes (0.444 mg mL⁻¹ siRNA, 45 μL, N, P ratio = 32), (f) percentage ITCH protein expression in rat brains (mean ± s.d., n = 5 and representative of 2 separate experiments) following the nasal administration of 4APPA-siRNA (0.9 mg mL⁻¹ siRNA, 45 μL, N, P ratio = 32 in phosphate buffer—2 mM), siRNA (0.9 mg mL⁻¹ siRNA, 45 μL), scrambled siRNA (0.89 mg mL⁻¹ siRNA, 45 μL) and phosphate buffer (2 mM) to male Sprague Dawley rats dosed twice daily for 3 days and killed 18 h after the last dose. * = statistically significantly different when compared to all other formulations (p < 0.05), ** = statistically significantly different when compared to all other formulations (p < 0.0001).