Intranasal brain delivery of cationic nanoemulsion-encapsulated TNFα siRNA in prevention of experimental neuroinflammation

Abstract

Neuroinflammation is a hallmark of acute and chronic neurodegenerative disorders. The main aim of this study was to evaluate the therapeutic efficacy of intranasal cationic nanoemulsion encapsulating an anti-TNFα siRNA, for potential anti-inflammatory therapy. TNFα siRNA nanoemulsions were prepared and characterized for particle size, surface charge, morphology, and stability and encapsulation efficiency. Qualitative and quantitative intracellular uptake studies by confocal imaging and flow cytometry, respectively, showed higher uptake compared to Lipofectamine® transfected siRNA. Nanoemulsion significantly lowered TNFα levels in LPS-stimulated cells. Upon intranasal delivery of cationic nanoemulsions almost 5 fold higher uptake was observed in the rat brain compared to non-encapsulated siRNA. More importantly, intranasal delivery of TNFα siRNA nanoemulsions in vivo markedly reduced the unregulated levels of TNFα in an LPS-induced model of neuroinflammation. These results indicate that intranasal delivery of cationic nanoemulsions encapsulating TNFα siRNA offered an efficient means of gene knockdown and this approach has significant potential in prevention of neuroinflammation.

From the clinical editor: Neuroinflammation is often seen in patients with neurodegenerative disorders and tumor necrosis factor-alpha (TNFα) plays a significant role in contributing to neuronal dysfunction. As a result, inhibition of TNFα may alleviate disease severity. In this article, the authors investigated using a cationic nanoemulsion system carrying TNFα siRNA intra-nasally to protect against neuroinflammation. This new method may provide a future approach in this clinical setting.

Keywords: Brain delivery; Cationic nanoemulsion; Gene silencing; Neuroinflammation; Small interfering RNA; Tumor necrosis factor-alpha.

Figure 1

Electrophoretic retardation analysis of TNFα silencing siRNA encapsulation in cationic nanoemulsion (SNE). The release of intact siRNA by 10% Triton® X100 was shown for SNE in lane 11(1A) Lane 1:Empty, Lane 2: siRNA standard 250ug/mL, Lane 3: siRNA standard 125ug/mL, Lane 4: siRNA standard 62.5ug/mL, Lane 5: siRNA standard 31.25ug/mL, Lane 6 to Lane 8: Empty, Lane 9: siRNA nanoemulsion (SNE) 250ug/mL with no Triton® X100, Lane 10: SNE 250ug/mL with 2% Triton, Lane 11: SNE 250ug/mL with 10% Triton. Figure 1B shows the electrophoretic retardation analysis of siRNA stability when formulated as siRNA nanoemulsion; Lane 1: Empty, Lane 2: SNE incubated with RNAse for 15min followed by PBS dilution, Lane 3: SNE incubated with RNAse followed by Triton® X100 treatment, Lane 4: SNE incubation with PBS and dilution with PBS, Lane 5: SNE incubated with Triton and PBS dilution, Lane 6: Empty, Lane 7: siRNA incubated with RNAse and diluted with PBS, Lane 8: siRNA incubated with RNAse and Triton treatment, Lane 9: siRNA diluted with PBS only, Lane 10: siRNA incubated with PBS and treatment with Triton® X100.

Figure 2

The uptake and trafficking of siRNA-encapsulated cationic nanoemulsion (SNE) in J774A.1 cells. Fluorescence microscopy images showing the blue (nucleus), red (siRNA) and overlay images for siRNA complexed in Lipofectamine® as compared to SNE at 200 nM concentrations. The images were taken at 40× original magnification.

Figure 3

Flow cytometry results showing the FL-2 channel (585/42 emission) which was used to detect the cells containing CY3-label siRNA containing particles (A). Flow cytometry data presented as percent fluorescence intensity/uptake for siRNA alone (siRNA) and nanoemulsion (SNE) compared to control untreated cells (B).

Figure 4

Cytotoxicity of siRNA encapsulated cationic nanoemulsion (SNE) at 2 hours (A) and 19 hour time point (B) post-administration in J774A.1 macrophages. The cell viability of the untreated cells was considered to be 100%, and the values obtained in the rest of the treatment groups were normalized to control values and presented in percentage form. The values reported are mean ± SD (n = 8).

Figure 5

TNFα gene knockdown result in lipopolysaccharide (LPS) stimulated macrophages showing inhibition of TNFα mRNA expression in the J774A.1 macrophage cell line. The data was plotted by considering the TNFα expression as 100% in LPS stimulated cells. The values reported are mean ± SD (n = 3) with **p<0.01 showing significant difference between groups

Figure 6

Results showing comparison for the percent administered dose in the brain (B) region highlighted (A) and plasma (C) following siRNA dosed in solution (STNF) versus siRNA dosed in cationic nanoemulsion (SNE) formulation. Brain targeting ratio comparing the targeting efficiency of siRNA using either solution versus nanoemulsion is shown (D). The values reported are mean ± SEM (n = 3); *p < 0.05.

Figure 7

Inflammatory marker TNFα and iNOS specific mRNA results showing transcript expression in the substantia nigra region of rat brain. Graph comparing the pre-treatment with TNFα siRNA in solution (STNF) and siRNA-loaded cationic nanoemulsion (SNE). Data was plotted by considering the expression of control saline group as 100%. The values reported are mean ± SEM (n = 3); *p < 0.05.

Figure 8

Body weight measurements to determine safety/tolerability profile upon single intranasal administration of the control and siRNA encapsulated cationic nanoemulsion (SNE) (A). Histology of nasal respiratory, olfactory epithelium (A) and liver (C) after intranasal dosing of siRNA. The values are reported as percent change in body weight as a function of pre-treatment weight of rats.