Evaluation of Selenomethionine Entrapped in Nanoparticles for Oral Supplementation Using In Vitro, Ex Vivo and In Vivo Models

Abstract

Selenium methionine (SeMet) is an essential micronutrient required for normal body function and is associated with additional health benefits. However, oral administration of SeMet can be challenging due to its purported narrow therapeutic index, low oral bioavailability, and high susceptibility to oxidation. To address these issues, SeMet was entrapped in zein-coated nanoparticles made from chitosan using an ionic gelation formulation. The high stability of both the SeMet and selenomethionine nanoparticles (SeMet-NPs) was established using cultured human intestinal and liver epithelial cells, rat liver homogenates, and rat intestinal homogenates and lumen washes. Minimal cytotoxicity to Caco-2 and HepG2 cells was observed for SeMet and SeMet-NPs. Antioxidant properties of SeMet were revealed using a Reactive Oxygen Species (ROS) assay, based on the observation of a concentration-dependent reduction in the build-up of peroxides, hydroxides and hydroxyl radicals in Caco-2 cells exposed to SeMet (6.25-100 μM). The basal apparent permeability coefficient (P_app) of SeMet across isolated rat jejunal mucosae mounted in Ussing chambers was low, but the P_app was increased when presented in NP. SeMet had minimal effects on the electrogenic ion secretion of rat jejunal and colonic mucosae in Ussing chambers. Intra-jejunal injections of SeMet-NPs to rats yielded increased plasma levels of SeMet after 3 h for the SeMet-NPs compared to free SeMet. Overall, there is potential to further develop SeMet-NPs for oral supplementation due to the increased intestinal permeability, versus free SeMet, and the low potential for toxicity.

Keywords: cytotoxicity; intestinal drug transport; nanoparticles; nutraceuticals; oral delivery of micronutrients; selenium methionine.

Figure 1

Effect of SeMet on inhibition of induction of ROS in Caco-2 cells by TBHP (100 µM). One-way ANOVA with Dunnett’s multiple comparison post-test, * p < 0.05, ** p < 0.01 and *** p < 0.001, compared to controls. Each value represents the mean ± SEM in triplicate (n = 3), with 3 independent replicates.

Figure 2

Stability of (A) SeMet (5 mM); (B) Recombinant human insulin (250 µM) in rat liver homogenates (LH—White), gut homogenates (GH—Check) and gut washes (GW—Black) and PBS control (PBS—Grey) as measured by RP-HPLC. One-way ANOVA with Dunnett’s multiple comparison; ** p < 0.01, *** p < 0.001 and **** p < 0.0001, respectively, compared with analyte at 0 min. Each value represents the mean ± SEM, n = 3.

Figure 3

MTS assessment of SeMet, unloaded NPs and SeMet NPs exposed for (A) 4 h in Caco-2 cells and (B) 72 h in HepG2 cells. Triton^® X-100 (0.05%) was used as positive control and no treatment as negative control (untreated). One-Way ANOVA with Dunnett’s post-test *** p < 0.001, ** p < 0.01. Each value presented was normalised against untreated control and calculated from three separate experiments (n = 3), each of which included six replicates.

Figure 4

P_app of FD4 was measured across isolate jejunal and colonic mucosae. Effect of apical addition of 100 µM SeMet and SeMet NPs on P_app on jejunal mucosae with cumulative concentration of SeMet transported across the jejunum to the basolateral side over 120 min. Each value represents the mean ± EM with n = 3.

Figure 5

Representative H&E and Alcian Blue staining of rat jejunal and colonic mucosae in adapted horizontal diffusion chambers following a 2 h exposure with SeMet and SeMet-NPs compared to untreated tissue. Black arrows indicate erosion of epithelial layer. Horizontal bars in each panel = 100 µm.

Figure 6

Measurement of plasma SeMet concentration following jejunal instillation of SeMet, SeMet-NPs and saline. Bonferroni test *** p < 0.001, ** p < 0.01, * p < 0.05, SeMet NPs compared to SeMet. Each value represents the mean ± SEM, n = 5 independent experiments.

Figure 7

H&E and Alcian Blue and neutral red staining of mucosae 180 min after intra-jejunal instillations. Groups: PBS control, free SeMet (20 µg/kg), SeMet NP (20 µg/kg). Images captured using a light microscope. Goblet cells are indicated by ‘G’. Black arrows show epithelial sloughing. Horizontal bars on panels = 250 µm.