Oral TNF-α siRNA delivery via milk-derived exosomes for effective treatment of inflammatory bowel disease

Abstract

Oral administration facilitates the direct delivery of drugs to lesions within the small intestine and colon, making it an ideal approach for treating patients with inflammatory bowel disease. However, multiple physical barriers impede the delivery of oral RNA drugs through the gastrointestinal tract. Herein, we developed a novel oral siRNA delivery system that protects nucleic acids in extreme environments by employing exosomes derived from milk to encapsulate tumor necrosis factor-alpha (TNF-α) siRNA completely. The remarkable structural stability of milk-derived exosomes (M-Exos), as opposed to those from HEK293T cells, makes them exceptional siRNA carriers. Results demonstrate that milk exosomes loaded with TNF-α siRNA (M-Exo/siR) can effectively inhibit the expression of TNF-α-related inflammatory cytokines. Moreover, given that milk exosomes are composed of unique lipids with high bioavailability, orally administered M-Exo/siR effectively reach colonic tissues, leading to decreased TNF-α expression and successful alleviation of colitis symptoms in a dextran sulfate sodium-induced inflammatory bowel disease murine model. Hence, milk-derived exosomes carrying TNF-α siRNA can be effectively employed to treat inflammatory bowel disease. Indeed, using exosomes naturally derived from milk may shift the current paradigm of oral gene delivery, including siRNA.

Keywords: Inflammatory bowel disease; Milk-derived exosome; Oral gene delivery; TNF-α; siRNA.

Graphical abstract

Fig. 1

Preparation of M-Exo/siR and optimization of electroporation conditions for loading TNF-α siRNA into M-Exos. (A) Schematic of the colitis treatment process through oral administration of M-Exo/siR. (B) Comparative changes of morphology of exosomes according to electroporation conditions. (C) Loading efficiency of TNF-α siRNA into M-Exos according to voltage. The mixing ratio of M-Exos and siRNA is the same in all conditions. Data are mean ± SD (n = 9). (D) Loading efficiency according to the M-Exo and TNF-α ratio. The voltage, pulse number, and pulse length used for electroporation are consistent in all conditions. Data are mean ± SD (n = 5). (E) Representative SRM images were measured in the x-, y-, and z-axis directions of M-Exo/siR. Red (Cyanine 5.5-labeled M-Exos); Green (5′-Fluorescein phosphoramidite-labeled TNF-α siRNA).

Fig. 2

Cytotoxicity and anti-inflammatory efficacy of M-Exo/siR in vitro. (A) Intracellular TNF-α siRNA delivery via M-Exo/siR in NCM460 cells. Blue (Hoechst 33342 fluorescence staining of nuclei); Green (5′-Fluorescein phosphoramidite-labeled TNF-α siRNA); Red (Cyanine 5.5-labeled M-Exos). (B) Cytotoxicity of M-Exo/siR in NCM460. Data are mean ± SD (n = 5); ns = not significant, **p < 0.01. (C) Comparison of NCM460 cell viability according to the concentration of M-Exo/siR and Exo-Fect™. Data are mean ± SD (n = 4); ***p < 0.001, ****p < 0.0001. (D) TNF-α protein down-regulation effect of M-Exo/siR in RAW 264.7 cells. Each group was treated with 100 ng/mL LPS for 8 h, followed by a 24 h incubation with their respective substances. Data are mean ± SD (n = 3); ***p < 0.001, ****p < 0.0001. (E) Relative mRNA expression of pro-inflammatory cytokines. Each group was treated with 100 ng/mL LPS for 8 h, followed by a 24 h incubation with their respective substances. Data are mean ± SD (n = 5); ***p < 0.001, ****p < 0.0001.

Fig. 3

In vivo therapeutic effect of intraperitoneally (i.p.) injected M-Exo/siR against colitis. (A) Treatment schedule of dextran sulfate sodium (DSS) colitis induction and i.p. injection of M-Exo/siR. After the acclimatization period, 8-week-old female BALB/c mice were i.p. injected with M-Exo/siR four times at three-day intervals beginning one day before 2.5 % DSS administration. (B) Daily body weight loss of mice. Data are mean ± SD (n = 8); ns = not significant, **p < 0.01, ****p < 0.0001. (C) Daily disease activity index (DAI) score. Data are mean ± SD (n = 8); ns = not significant, **p < 0.01, ****p < 0.0001. (D) Representative extracted colon image and colon length on day 28. Data are mean ± SD (n = 8); ns = not significant, *p < 0.05, ***p < 0.001. (E) Serum TNF-α cytokine level on day 28. Data are mean ± SD (n = 4); ns = not significant, **p < 0.01, ****p < 0.0001. (F) Tnfa mRNA level in colitis colon tissue on day 28. Data are mean ± SD (n = 4); ns = not significant, **p < 0.01. (G) Representative histopathological images of colon tissues stained with hematoxylin and eosin (H&E) on day 28. Open black circles (loss of surface epithelium); black arrows (infiltration of inflammatory cells); black triangles (ulceration); open black triangles (loss of goblet cells).

Fig. 4

Stability and lipid composition analysis of M-Exos and H-Exos. (A) Bio-distribution of Cy5.5-labeled M-Exos. After oral administration of saline, Cyanine 5.5, Cyanine 5.5-labeled H-Exos, and Cyanine 5.5-labeled M-Exos to 8-week-old female BALB/c, radiant efficiency was measured at 8 h, 24 h, 48 h, or 72 h. (B) Tissue images of Cy3-labeled siRNA absorbed into the stomach, small intestine, and colon. Green (Cyanine 3-labeled TNF-α siRNA). (C) Lipidomics analysis of M-Exos compared to H-Exos. Data are mean ± SD (n = 4). (D) Lipid composition ratio constituting H-Exos and M-Exos.

Fig. 5

In vivo colitis treatment efficacy of M-Exo/siR through oral gavage. (A) Treatment schedule of DSS colitis induction and oral administration of M-Exo/siR. After the acclimatization period, 8-week-old female BALB/c mice were orally administered M-Exo/siR four times at three-day intervals beginning one day before 2.5 % DSS administration. (B) Daily body weight loss of mice. Data are mean ± SD (n = 10); ****p < 0.0001. (C) Daily disease activity index (DAI) score. Data are mean ± SD (n = 10); ****p < 0.0001. (D) Representative extracted colon image and colon length on day 28. Data are mean ± SD (n = 8); ***p < 0.001 and ****p < 0.0001. (E) Serum TNF-α cytokine level on day 28. Data are mean ± SD (n = 4); ns = not significant, **p < 0.01, ***p < 0.001 (F) Tnfa mRNA level in colitis colon tissue on day 28. Data are mean ± SD (n = 4); *p < 0.05, ***p < 0.001, ****p < 0.0001. (G) Representative histopathological images of colon tissues stained with hematoxylin and eosin (H&E) on day 28. Open black circles (loss of surface epithelium); black arrows (infiltration of inflammatory cells); black triangles (ulceration); open black triangles (loss of goblet cells). (H) Analysis of inflammatory cytokine mRNA expression in colonic tissues. Data are mean ± SD (n = 4); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (I) Colonic myeloperoxidase (MPO) activity. Data are mean ± SD (n = 6); ****p < 0.0001.