Temperature-triggered in situ forming lipid mesophase gel for local treatment of ulcerative colitis

Abstract

Ulcerative colitis is a chronic inflammatory bowel disease that strongly affects patient quality of life. Side effects of current therapies necessitate new treatment strategies that maximise the drug concentration at the site of inflammation, while minimizing systemic exposure. Capitalizing on the biocompatible and biodegradable structure of lipid mesophases, we present a temperature-triggered in situ forming lipid gel for topical treatment of colitis. We show that the gel is versatile and can host and release drugs of different polarities, including tofacitinib and tacrolimus, in a sustained manner. Further, we demonstrate its adherence to the colonic wall for at least 6 h, thus preventing leakage and improving drug bioavailability. Importantly, we find that loading known colitis treatment drugs into the temperature-triggered gel improves animal health in two mouse models of acute colitis. Overall, our temperature-triggered gel may prove beneficial in ameliorating colitis and decreasing adverse effects associated with systemic application of immunosuppressive treatments.

Fig. 1. In vitro characterization of the TIF-Gel.

a schematic depiction of the in vitro characterization and the mechanism of the gel formation. b SAXS spectra acquired at different temperatures (25, 30 and 38 °C; bottom, middle and top spectra, respectively) on gels containing increasing amounts of water (12%, 14%, 16% and 18 % w/w) and c SAXS spectra acquired at different times (5, 10, 20 and 30 min) after incubation at 38 °C; d frequency sweep at the end (purple symbols) and beginning (grey symbols) of the release experiments. e Flow and yield points obtained for the lamellar phase (grey bars) and cubic gel (purple bars). f SAXS before (1) and after incubation of LMPs in HEPES buffer (2) and in HEPES buffer enriched with 1000 U/mL of lipase (3). The LMP graphics (L; cubic ia3d, cubic pn3m and hexagonal) are adapted from ref. with the permission of AIP (10.1063/PT.3.4522) and from ref. with permission of RSC (10.1039/D2TB00403H). Additional graphics (Franz cell, gel and colon tract) were created using BioRender. Source data are provided as a source data file.

Fig. 2. In vitro and ex vivo characterizations of the drug-loaded TIF-Gel.

a TOFA-loaded gel (TIF-Gel-TOFA) SAXS spectra acquired at different temperatures; b in vitro release of free drug (TOFA, blue line) and TIF-Gel-TOFA in HEPES buffer (black line) and in the presence of lipase (green line); c ex vivo release of free drug (TOFA, blue line) and TIF-Gel-TOFA (black line); d TAC loaded LMPs (TIF-Gel-TAC) SAXS spectra acquired at different temperatures; e in vitro release of free drug (TAC, blue line), TIF-Gel-TAC (black line) and in the presence of lipase (green line); f ex vivo release of free drug (TAC, blue line) and TIF-Gel-TAC in HEPES buffer (black line). Results in (b, c, e, f) are reported as mean ± STDV (n = 3). Cartoons (Franz cell, gel and X-ray pattern) are created by BioRender. Source data are provided as a source data file.

Fig. 3. TIF-Gel-TOFA effectively mitigates intestinal inflammation and disease induced by DSS treatment in mice.

Mice were prophylactically treated rectally with either empty gel (TIF-Gel; n = 6), tofacitinib in vehicle (TOFA; n = 7), or TOFA loaded-gel (TIF-Gel-TOFA; n = 6) and thereafter challenged with 2% DSS in the drinking water. Treatments were then applied every other day until the end of the experiment. Weights (a) and disease score (b) were recorded throughout the experiment. At the end of the experiment, spleens, mesenteric lymph nodes (mLNs) and colons were removed from the mice. The spleens were weighed (c) and single splenocytes were enumerated (d). The tissue concentrations of various cytokines were measured (e). The mouse colon length was measured (f), and the colon was opened transversally, cleaned, and prepared for histology (g). Colon histopathology scores were determined by a blinded pathologist and aggregated (h). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and actual value is provided for values less than 0.1 but not meeting significance threshold as determined by two-way ANOVA (a), multiple Student’s t tests with Holm–Sidak correction for multiple comparisons (b), and one way ANOVA with multiple comparisons and Tukey correction (c, d, e, f, h). All tests were performed using Prism (GraphPad) and applying default settings for the above-mentioned analyses; naive values were excluded from analyses; all error bars are ± SEM. Source data are provided as a source data file.

Fig. 4. Assessment of the effect of TAC-loaded TIF-Gel on T cell-mediated colitis.

12–15-week-old Rag^−/− mice develop colitis via transfer of 2.5 × 10⁵ naive CD4 + T cells. Starting on day 3 after T cell transfer, mice (n = 6) received daily rectal instillations with TIF-Gel without drug (TIF-Gel), TAC-loaded TIF-Gels (TIF-Gel-TAC) or TAC in vehicle (TAC). a Schematic overview on the experimental set-up; b weight development over the course of the experiment; c cumulative disease activity score; d colon length and spleen weight; e representative pictures and respective scoring from mouse colonoscopy on day 19 post T cell transfer and from H&E-stained sections of the terminal colon collected on day 19 post T cell transfer. *P < 0.05, **P < 0.01 as determined by two-way ANOVA (b, c) and one way ANOVA with multiple comparisons and Tukey correction (d, e). All tests were performed using Prism (GraphPad) and applying default settings for the above-mentioned analyses; all error bars are ±SEM. Source data are provided as a source data file.

Fig. 5. Immune cell populations and cytokine levels in the colon from TAC-loaded TIF-Gel (TIF-Gel-TAC) treated mice.

12–15-week-old Rag^−/− mice develop colitis via transfer of 2.5 × 10⁵ naive T cells. Starting on day 3 post T cell transfer, mice received daily rectal instillations with TIF-Gel without drug (TIF-Gel), TAC-loaded TIF-Gels (TIF-Gel-TAC) or TAC in vehicle (TAC). Depicted are the relative abundance of the indicated cell populations in (a) the colonic lamina propria, b mesenteric lymph nodes; and c the spleen on day 19 after T cell transfer, and d levels of indicated cytokines in colon lysates. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by one way ANOVA with multiple comparisons and Tukey correction. All tests were performed using Prism (GraphPad) and applying default settings for the above-mentioned analyses; all error bars are ±SEM. Source data are provided as a source data file.

Fig. 6. Drug delivery via TIF-Gel leads to a low systemic drug exposure.

a Experimental design for the pharmacokinetic study. Healthy mice (n = 5/group) received a single enema of either drug-loaded TIF-Gel (TIF-Gel-TOFA or TIF-Gel-TAC) or free drugs (TOFA or TAC). The plasma drug concentrations were measured at the indicated time points after administration. Plasma concentration versus time profiles of the pharmacokinetic experiment of TOFA- (b) and TAC-treated animals (c) and Area Under the Curve (AUC)_0-48h values of TOFA- and TAC-treated mice (d, e, respectively). ***P < 0.001 as determined by two tailed Student’s t test. All tests were performed using Prism (GraphPad); all error bars are ±SD. Source data are provided as a source data file.