Garcinol Encapsulated Ph-Sensitive Biodegradable Nanoparticles: A Novel Therapeutic Strategy for the Treatment of Inflammatory Bowel Disease

Abstract

The emergence of pH-sensitive nanoscale particles is beneficial due to their ability to only release cargo in a colonic pH environment, which helps to directly target inflamed tissues in inflammatory bowel disease (IBD). Hence, we have designed the formulation of pH-sensitive biodegradable garcinol (GAR)-loaded poly (lactic-co-glycolic acid) (PLGA) coated with Eudragit^® S100 (ES100) (GAR-PLGA-ES100 nanoparticles (NPs)) for reducing inflammation caused by proinflammatory cytokines. The GAR-PLGA-ES100 NPs were prepared using a solvent evaporation technique and characterized for shape and surface morphology. An in vitro drug release study revealed the release of the drug specifically from NPs at the colonic pH of 7.4. The in vitro cytotoxicity of the GAR-PLGA-ES100 NPs was also evaluated and found to be highly biocompatible with CACO-2 cells. These NPs were able to reduce lactate dehydrogenase (LDH) and myeloperoxidase (MPO) activity. Inhibition of the expression of pro-inflammatory cytokine TNF-α , chemokine interleukin (IL)-8 and the nuclear factor kappa light chain enhancer of activated B-cells (NF-κB) was observed after GAR-PLGA-ES100 NPs treatment. Therefore, our results support the idea that GAR-PLGA-ES100 NPs show substantial improvement after the release of the drug, specifically in colonic pH targeting and reduction in the activation of inflammation that leads to IBD, suggesting that GAR-PLGA-ES100 NPs are promising candidates for oral delivery to colonic inflamed tissue.

Keywords: eudragit S100; garcinol; inflammatory bowel disease; nuclear factor kappa light chain enhancer of activated B-cells; pH-sensitive nanoparticles.

Figure 1

Size distribution of GAR-PLGA-ES100 NPs by DLS measurement.

Figure 2

SEM image of GAR-PLGA-ES100 NPs (At scale 3.00 μm).

Figure 3

(a) In vitro release of GAR from GAR-PLGA-ES100 NPs and GAR-PLGA NPs in phosphate buffered saline (PBS) (pH 1.2, PBS/FBS buffer) studied over 2 days at 37 °C. (b): In vitro release of GAR from GAR-PLGA-ES100 NPs and GAR-PLGA NPs in PBS (pH 5.6, PBS/FBS buffer) studied over 2 days at 37 °C. (c): In vitro release of GAR from GAR-PLGA-ES100 NPs and GAR-PLGA NPs in PBS (pH 7.4 PBS/FBS buffer) studied over 2 days at 37 °C.

Figure 3

(a) In vitro release of GAR from GAR-PLGA-ES100 NPs and GAR-PLGA NPs in phosphate buffered saline (PBS) (pH 1.2, PBS/FBS buffer) studied over 2 days at 37 °C. (b): In vitro release of GAR from GAR-PLGA-ES100 NPs and GAR-PLGA NPs in PBS (pH 5.6, PBS/FBS buffer) studied over 2 days at 37 °C. (c): In vitro release of GAR from GAR-PLGA-ES100 NPs and GAR-PLGA NPs in PBS (pH 7.4 PBS/FBS buffer) studied over 2 days at 37 °C.

Figure 4

Cellular uptake of Coumarin-6 PLGA-ES100 NPs in CACO-2 cells by confocal microscopy; untreated CACO-2 cells (a–d), CACO-2 cells treated with Coumarin-6 PLGA-ES100 NPs for 4 h (e–h) (on a scale of 25 μm).

Figure 5

In vitro cytotoxicity assay on CACO-2 cells for 24–48 h. blank-PLGA-ES100 NPs concentration was 500 μg/mL, free GAR was 50 μg/mL and GAR-PLGA-ES100 NPs (50, 100, 250, and 500 μg/mL). Student’s unpaired t-test was carried out to check the statistical significance of the experiment (ns: not significant; * p < 0.05).

Figure 6

Lactate dehydrogenase (LDH) assay on CACO-2 cells treated for 24 h with blank-PLGA-ES100 NPs. Concentration was 500 μg/mL, free GAR was 50 μg/mL and GAR-PLGA-ES100 NPs (50, 100, and 250 μg/mL). Student’s unpaired t-test was carried out to check the statistical significance of the experiment (ns: not significant; * p < 0.05).

Figure 7

Myeloperoxidase (MPO) assay on CACO-2 cells treated for 24 h with blank-PLGA-ES100 NPs. Concentration was 500 μg/mL, free GAR was 50 μg/mL and GAR-PLGA-ES100 NPs (50, 100, and 250 μg/mL). Student’s unpaired t-test was carried out to check the statistical significance of the experiment (ns: not significant; * p < 0.05).

Figure 8

Induction and inhibition of inflammation in CACO-2 cells by confocal microscopy: non-inflamed CACO2 cells (a–d); inflamed CACO-2 cells treated with proinflammatory cocktail comprising of IL-6, TNF-α, and LPS at 0.2, 0.3, and 20 μg/mL, respectively, for 3 h used to observe the expression of NF-κB (e–h). Reduction in the expression of NF-κB occured in CACO-2 cells after treatment with GAR-PLGA-ES100 NPs (250 μg/mL) for 48 h (i–l) (at scale 25 μm).

Figure 9

Induction and inhibition of inflammation in CACO-2 cells by confocal microscopy: non-inflamed CACO2 cells (a–d); inflamed CACO-2 cells treated with proinflammatory cocktail comprising of IL-6, TNF-α, and LPS at 0.4, 0.9, and 30 μg/mL, respectively, for 3 h used to observe the expression of TNF- α (e–h). Reduction in the expression of TNF- α occurred in CACO-2 cells after treatment with GAR-PLGA-ES100 NPs (250 μg/mL) for 48 h (i–l) (at scale 25 μm).

Figure 10

Induction and inhibition of inflammation in CACO-2 cells by confocal microscopy: non-inflamed CACO2 cells (a–d); CACO-2 cells treated with proinflammatory cocktail comprising of IL-6, TNF-α, IL-1β, and LPS at 0.4, 0.9, 1, and 30 μg/mL, respectively, for 45 min demonstrated the expression of IL-8 (e–h). Reduction in the expression of IL-8 in CACO-2 cells was observed after treatment with GAR-PLGA-ES100 NPs (250 μg/mL) for 48 h (i–l) (at scale 25 μm).