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