Attenuation of Chronic Inflammation in Intestinal Organoids with Graphene Oxide-Mediated Tumor Necrosis Factor-α_Small Interfering RNA Delivery

Abstract

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with a complex and multifactorial etiology, making it challenging to treat. While recent advances in immunomodulatory biologics, such as antitumor necrosis factor-α (TNF-α) antibodies, have shown moderate success, systemic administration of antibody therapeutics may lead to several adverse effects, including the risk of autoimmune disorders due to systemic cytokine depletion. Transient RNA interference using exogenous short interfering RNA (siRNA) to regulate target gene expression at the transcript level offers an alternative to systemic immunomodulation. However, siRNAs are susceptible to premature degradation and have poor cellular uptake. Graphene oxide (GO) nanoparticles have been shown to be effective nanocarriers for biologics due to their reduced cytotoxicity and enhanced bioavailability. In this study, we evaluate the therapeutic efficacy of GO mediated TNF-α_siRNA using in vitro models of chronic inflammation generated by treating murine small intestines (enteroids) and large intestines (colonoids) with inflammatory agents IL-1β, TNF-α, and LPS. The organotypic mouse enteroids and colonoids developed an inflammatory phenotype similar to that of IBD, characterized by impaired epithelial homeostasis and an increased production of inflammatory cytokines such as TNF-α, IL-1β, and IL-6. We assessed siRNA delivery to these inflamed organoids using three different GO formulations. Out of the three, small-sized GO with polymer and dendrimer modifications (smGO) demonstrated the highest transfection efficiency, which led to the downregulation of inflammatory cytokines, indicating an attenuation of the inflammatory phenotype. Moreover, the transfection efficiency and inflammation-ameliorating effects could be further enhanced by increasing the TNF-α_siRNA/smGO ratio from 1:1 to 3:1. Overall, the results of this study demonstrate that ex vivo organoids with disease-specific phenotypes are invaluable models for assessing the therapeutic potential of nanocarrier-mediated drug and biologic delivery systems.

Figure 1

Intestinal organoids show organ specific cell types and morphology. (A) Bright-field images of enteroids and colonoids at day 5 of culture. Scale bars measure 500 μm. Immunofluorescence images of day 10 (B) enteroids and (C) colonoids. Lgr5 (red), E-cadherin (red), villin (green), ChgA (green), and Muc2 (red) show the presence of epithelial stem cells, enterocytes, goblet cells, and enteroendocrine cells. Paneth cells are shown with lysozyme (green) in panel (B) and nucleus (Hoechst, blue) in panels (B) and (C). Scale bars measure 500 μm.

Figure 2

Chemical modification of graphene oxide. AFM topography images of (A) sGO, (B) smGO, (C) zoomed-in smGO, and (D) bmGO demonstrate single-layer GO flakes. (E) FT-IR spectra of sGO, smGO, and bmGO show functional groups such as hydroxyl (O–H), carbonyl (C=O), and carbon–carbon double bonds (C=C) on GO reacting with PEG and PAMAM and with decreased absorbance intensity on smGO and bmGO. New functional groups such as amide groups and sharp C–O were evidently observed on smGO and bmGO.

Figure 3

Increasing concentrations (0.25, 1, 4, and 16 μg/mL) of GO formulations impair viability. Relative viability of (A) enteroids and (B) colonoids determined using WST-8 assay. Values on each graph are shown as mean ± SD of three independent experiments each with replicates (n = 3). Statistical significance was determined with one-way ANOVA. P < 0.05 was considered significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. P > 0.05, ns: not significant.

Figure 4

Treatment with proinflammatory factors gave rise to proinflammatory phenotypes. Brightfield images of (A) enteroids and (B) colonoids after 6 days of induction. Scale bars measure 500 μm. (C) Relative viability of intestinal organoids was determined using the WST-8 assay. (D) Immunofluorescence images of intestinal organoids showing TUNEL^+ve apoptotic cells (Hoechst, blue and TUNEL, red). Top panel: enteroids. Scale bars measure 250 μm. Bottom panel: colonoids. Scale bars measure 100 μm. (E) Relative fold change of mRNA expression for TNF-α after 7 days of induction. Concentrations of (F) TNF-α, (G) IL-1β, and (H) IL-6. Values on each graph are shown as mean ± SD of six independent experiments (n = 6). Statistical significance was determined with t test (for comparing two groups) and one-way ANOVA (for comparing three groups or more). P > 0.05 was considered not significant (ns). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 5

The highest degree of transfection was demonstrated by smGO. (A) Knockdown efficiency for TNF-α after 48 h of transfections. Increased knockdown efficiency translated to reduced cytokine secretion of (B) TNF-α, (C) IL-1β, and (D) IL-6. (E) Knockdown efficiency for TNF-α after transfections of enteroids and colonoids with 3:1 siRNA:smGO. Increased knockdown efficiency with the 3:1 formulation resulted in further reduction of cytokine secretion for (F) TNF-α, (G) IL-1β, and (H) IL-6. Values on each graph are shown as mean ± SD of six independent experiments (n = 6). Statistical significance was determined with t test (for comparing two groups) and one-way ANOVA (for comparing three groups or more). P > 0.05 was considered ns: not significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.