Abstract
NF‐κB, a critical cytokine of inflammatory bowel diseases (IBD), is a viable marker to reflect the inflammatory activity of the intestine. We aimed to develop NF‐κB‐targeted microbubbles (MBs) and perform molecular contrast‐enhanced ultrasound (CEUS) to quantify NF‐κB expressions on the intestinal wall in IBD mice in vivo. In this study, NF‐κB‐targeted MBs were fabricated by connecting biotin‐loaded NF‐κB antibodies and avidin‐loaded MBs. NF‐κB‐targeted MBs presented as transparent and round bubbles with an average diameter of 1.03/μm±0.01. The specific binding of targeted MBs and inflammatory cells was validated by in vitro experiments, including flow cytometry, Western blot and immunofluorescence, which revealed the specific binding of targeted MBs and inflammatory cells. Subsequently, NF‐κB‐targeted CEUS imaging was performed on mice with chemical‐induced colitis, and the peak intensity (PI) and time‐to‐peak (TTP) were quantified. Pathological and immunohistochemical (IHC) examinations were further implemented. For the target CEUS group, fast enhancement followed by slow subsiding was observed. The PI of target CEUS of the IBD mice was significantly higher than that of non‐target CEUS of the IBD mice, healthy controls and target CEUS of the treated IBD mice (34835%[13379–73492%] VS 437%[236–901%], 130%[79–231%], 528%[274–779%], p<0.0001), in accordance with the IHC results of NF‐κB expressions. The TTP of target CEUS of the treated mice was significantly higher than that of untreated mice (35.7s [18.1–49.5s] VS 8.3s [4.2–12.5s], p<0.0001). Therefore, we suggested that NF‐κB‐targeted CEUS could accurately detect and quantify NF‐κB expressions on the intestinal walls of IBD, enabling the evaluation of intestinal inflammation.
Highlights
Studies have shown that the expression level of NF-κB on intestinal epithelial cells is highly associated with the disease activity, and indicative of the treatment response and the risk of developing colitis-associated cancer.12-14 an accurate assessment of NF-κB expressions in vivo can be helpful in guiding the management of inflammatory bowel diseases (IBD)
NF-κB-targeted MBs were fabricated by connecting NF-κB-p65 Abs onto the surface of MBs, and the MBs were proved be and effectively conjugated with the inflammatory cells in vitro with high expressions of NF-κB-p65
Targeted contrast-enhanced ultrasound (CEUS) was performed on dextran sodium sulphate (DSS)-induced IBD mice, and a pattern of fast enhancement and slow subsiding was observed in target CEUS group of IBD mice
Summary
Inflammatory bowel diseases (IBD) are systematic, chronic inflammatory diseases, including ulcerative colitis (UC) and Crohn disease (CD).[1,2] The disease often begins in adolescence and has a high recurrence rate, causing a huge economic and social burden. After incubated at room temperature for 30 minutes, the mixture was added with dihydrochloride (DAPI) (Origene Biotechnology Co., Ltd, Wuxi, China) staining solution It was washed with PBS for 3 times and put on the glass side for observing under the fluorescence microscope. The confocal laser scanning microscopy (Nikon Ti-E+C2, Japan) was performed to observe the intracellular distributions of the FITC- NF-κB-targeted MBs. For DAPI microscopic imaging, the excitation wavelength was 358 nm, and the emission wavelength was 461 nm. The IHC examination was performed according to the protocol presented by Mori H et al.[21] The samples were incubated with rabbit anti-NF-κB-p65 antibodies, biotinylated goat anti-rabbit IgG and Streptavidin Alexa Flour 546 conjugate (Abcam, ab16502, Aobsen biochem, Shanghai, China). A p value of
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