Edinburgh college's consensus statements are not purely for UK
Authors: M. Dennis, M. Farquhar, P. Langhorne, G. Lowe, C. Warlow
Journal: BMJ (1999)

<b>9</b> <h3><b>Purpose:</b></h3> Inflammatory bowel disease (IBD) is characterized by chronic relapsing immune mediated inflammation in the gastrointestinal tract, and hydrogen sulfide (H<sub>2</sub>S) is one of the emerging therapeutic targets of IBD because of its anti-inflammatory functions. We hypothesized that spleen targeting H<sub>2</sub>S donating liposome (H<sub>2</sub>S-lipo) can alleviate the severity of dextran sodium sulfate induced colitis (DSS-colitis, a disease model of IBD) because spleen is the largest lymphoid organ that has ability of peripheral T<sub>reg</sub> differentiation (TRD) and immune modulation. <h3><b>Methods:</b></h3> H<sub>2</sub>S-lipo was synthesized using phospholipids (PS), PS-polyethylene glycol (PS-PEG), cholesterol and a H<sub>2</sub>S donor, GYY 4137 (GYY). The H<sub>2</sub>S-lipo were synthesized using PS:PS-PEG ratios of 12:1, 6:1, 2:1 and 1:1 [H<sub>2</sub>S-lipo(12:1), H<sub>2</sub>S-lipo(6:1), H<sub>2</sub>S-lipo(2:1), H<sub>2</sub>S-lipo(1:1)] . Size distribution was observed up to 14 days for stability test in pure water at 25 ℃. H<sub>2</sub>S donor loading test used silver nitrate precipitation which could be detected at 405 nm. Spleen uptake difference were analyzed by PET imaging using <sup>64</sup>Cu labeled H<sub>2</sub>S-lipo. Anti-inflammatory M2 macrophage differentiation (M2D) was tested. Cell viability were observed MTT assay. Cell uptake was identified using fluorescent H2S lipo at Raw 264.7. H<sub>2</sub>S release was identified after uptake using H<sub>2</sub>S detection probe Hsip-1. M2D by released H<sub>2</sub>S was measured using the elongation factor (<i>1</i>) <b>(FigS H)</b>. DSS-colitis was induced by 3% DSS water <b>(Fig D)</b>. The test materials which are saline, GYY4137, and H<sub>2</sub>S-lipo were injected intravenously every day. The weight change was calculated for observing therapeutic effect. After sacrificed, whole colon length and colon weight per distal 3 cm were calculated (<i>2</i>). Inflammation score of H&amp;E staining images were compared (<i>3</i>). The TRD of extracted DSS-colitis spleen and bone marrow were analyzed by flow cytometry. <h3><b>Results:</b></h3> The sizes of H<sub>2</sub>S-lipo (6:1), H<sub>2</sub>S-lipo (2:1), and H<sub>2</sub>S-lipo (1:1) were stable for 14 days but that of H<sub>2</sub>S-lipo (12:1) was not <b>(FigS A-B)</b>. H<sub>2</sub>S donor loading efficiency was the highest in the H<sub>2</sub>S-lipo (6:1) <b>(FigS C)</b>. Also, in in vivo PET imaging after injection of the <sup>64</sup>Cu labeled H<sub>2</sub>S-lipo, the spleen/muscle ratio was the highest in H<sub>2</sub>S-lipo (6:1) injected mice <b>(Fig B-C)</b>. Note that H<sub>2</sub>S-lipo (12:1) was not used in the in vivo experiments because of its low stability. Therefore, H<sub>2</sub>S-lipo (6:1) is used and written as H<sub>2</sub>S-lipo in the further experiments. Effective cellular uptake of H<sub>2</sub>S-lipo was confirmed in Raw 264.7 cells <b>(FigS E)</b>. H<sub>2</sub>S-lipo showed significantly higher H<sub>2</sub>S production than GYY or saline in Raw 264.7 cells<b> (Fig A, FigS G)</b>. Furthermore, H<sub>2</sub>S-lipo was more effective in M2 macrophage differentiation than liposome or GYY <b>(Fig S F, I)</b>. In DSS colitis treatment experiment, the weight change of mice injected with H<sub>2</sub>S-lipo (100 µM) is significantly smaller than saline injected mice <b>(Fig G)</b>. In histologic analysis of intestine, inflammation score of H<sub>2</sub>S-lipo injected group was significantly lower than those of other groups <b>(Fig E, FigS J)</b>. Colon contraction and colon weight of H<sub>2</sub>S-lipo is significantly lower than other groups <b>(Fig F)</b>. Finally, TRD of H<sub>2</sub>S-lipo is significantly higher than other groups in DSS-colitis spleen <b>(FigS K,M)</b> and bone marrow <b>(FigS L,N)</b>. <h3><b>Conclusions:</b></h3> We developed H<sub>2</sub>S donating liposome which has spleen targeting and immune modulatory abilities<b>.</b> H<sub>2</sub>S-lipo demonstrated a therapeutic potential in DSS induced colitis model.