Inhibition of Angiogenesis and Effect on Inflammatory Bowel Disease of Ginsenoside Rg3-Loaded Thermosensitive Hydrogel

Abstract

Background: Inflammatory bowel disease (IBD), characterized by chronic inflammation of the digestive tract, involves angiogenesis as a key pathogenic mechanism. Ginsenoside Rg3, derived from the traditional Chinese herb ginseng, is recognized for its anti-angiogenic properties but is limited by low oral bioavailability. This necessitates the development of an alternative delivery system to improve its therapeutic effectiveness. Methods: Pluronic F-127 (F127) and Pluronic F-68 (F68) were used to construct Rg3-loaded thermosensitive hydrogel Gel-Rg3. Meanwhile, a series of physicochemical properties were determined. Then the safety and pharmacological activity of Gel-Rg3 were evaluated in vitro and in vivo using human umbilical vein endothelial cells (HUVECs) and colitis mouse model, in order to initially validate the potential of Gel-Rg3 for the treatment of IBD. Results: We engineered a rectally administrable, thermosensitive Gel-Rg3 hydrogel using F127 and F68, which forms at body temperature, enhancing Rg3's intestinal retention and slowly releasing the drug. In vitro, Gel-Rg3 demonstrated superior anti-angiogenic activity by inhibiting HUVEC proliferation, migration, and tube formation. It also proved safer and better suited for IBD's delicate intestinal environment than unformulated Rg3. In vivo assessments confirmed increased intestinal adhesion and anti-angiogenic efficacy. Conclusions: The Gel-Rg3 hydrogel shows promise for IBD therapy by effectively inhibiting angiogenesis via rectal delivery, overcoming Rg3's bioavailability limitations with improved safety and efficacy. This study provides new inspiration and data support for the design of treatment strategies for IBD.

Keywords: angiogenesis; drug delivery system; ginsenoside Rg3; inflammatory bowel disease; thermosensitive hydrogel.

Scheme 1

A schematic representation of Gel-Rg3 being gelated at intestinal temperature, adhering to the bowel and slowly releasing the drug, eventually acting on angiogenesis.

Figure 1

The phase transition temperature (T_sol–gel) curve. (A) Gel–Blank with different ratio of F68 (%, w/v) and same 25% F127 (w/v), (B) Gel–Blank with different ratio of F127 (%, w/v) and same 7.5% F68 (w/v), (C) Gel-Rg3 with different ratio of F68 (%, w/v) and same 25% F127 (w/v), (D) Gel-Rg3 with different ratio of F127 (%, w/v) and same 12.5% F68 (w/v).

Figure 2

Characterization of hydrogels. (A) Morphology of Gel–Blank and Gel-Rg3. (B) FT–IR spectras of F127, F68, Gel–Blank, Rg3 and Gel-Rg3. (C,D) SEM image of F68/F127 hydrogel at different magnifications. (E) Cumulative release profile of Rg3 from the hydrogel at 37 °C in release medium.

Figure 3

Rheological properties of hydrogels. (A) Storage modulus (G′) and loss modulus (G″) of Gel–Blank with temperature. (B) Storage modulus (G′) and loss modulus (G″) of Gel-Rg3 with temperature. (C) Viscosity of Gel–Blank as a function of shear rate. (D) Viscosity of Gel-Rg3 as a function of shear rate.

Figure 4

In vitro cytotoxicity and cell proliferation of hydrogels. (A) Fluorescent images of live/dead staining (100×). (B) Results of the semiquantitative analysis. (C) Survival percentage of HUVECs after 24 h of co-incubation with Gel–Blank. (D) HUVECs were exposed to various concentrations of Rg3 for 24 and 48 h. (E) HUVECs were exposed to various concentrations of Gel-Rg3 for 24 and 48 h. Cytotoxicity was measured using CCK-8. Data were shown as mean ± SDs (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control.

Figure 5

In vitro anti-angiogenesis efficacy of Gel-Rg3. (A) Optical images of HUVECs scratching assays treated with or without Gel-Rg3 and Rg3 (40×). (B) HUVECs migration rate of Gel-Rg3 and Rg3. (C) Optical images of tube formation treated with or without different concentrations of Gel-Rg3 and Rg3 (100×). Data were shown as mean ± SDs (n = 3), * p < 0.05, *** p < 0.001, **** p < 0.0001 vs. control.

Figure 6

Retention of F68/F127 hydrogel in vivo. (A) Representative pictures of live imaging of isolated intestine were collected 2 h, 6 h, 10 h after rectal administration. (B) Representative fluorescence images of colon sections obtained from different points in time after administration. The red fluorescence represents ICG retained in the intestine. (C) A semiquantitative analysis of the mean fluorescence intensity. (D) Data were shown as mean ± SDs (n = 3), ** p < 0.01, **** p < 0.0001, ns > 0.1.

Figure 7

In vivo efficacy evaluation. (A) The representative pictures of the colon in different groups. (B) The lengths of the colons were measured. (C) Histopathological changes in the colon in each group were assessed using H&E staining. (D,E) Evaluation of CD31-labeled microvasculature in colon mucosa of different groups using immunofluorescence (IF) (bule fluorescence: DAPI; Red fluorescence: CD31) and immunohistochemistry (IHC); the stain area of CD31 was measured and is displayed in (F) IF and (G) IHC. Data were shown as mean ± SDs (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns > 0.01.