Impact of yeast cell wall incorporation on the mucoadhesion, stability, oral permeability and release profile of alginate/whey protein beads loaded with insulin

Abstract

This study aimed to evaluate the impact of incorporating yeast cell wall (YCW) into alginate/whey protein (ALG/WP) particles as a strategy to improve oral insulin delivery. Insulin-loaded particles were produced by an extrusion–gelation process with or without YCW, and their physicochemical, mucoadhesive, and permeability properties were assessed in vitro, ex vivo, and in vivo. The inclusion of YCW increased the viscosity of the polymeric solution, resulting in more cohesive particles and a significant reduction in insulin loss during coating. Encapsulation efficiencies ranged from 65 to 99%. However, YCW did not significantly affect particle size or the release mechanism, which remained diffusion-controlled. Although YCW-containing beads exhibited enzyme inhibitory and mucoadhesive properties, insulin protection against enzymatic degradation was similar to that of control beads. YCW moderately enhanced insulin permeability in Caco-2 cell monolayers without cytotoxicity, consistent with a reversible reduction of transepithelial electrical resistance. This effect did not translate into a measurable increase in insulin absorption in ex vivo duodenum or in vivo duodenal administration, indicating that its contribution as an absorption enhancer is limited under physiologically complex conditions.

Introduction

The oral route remains the most common and preferred route of drug administration because of its convenience, safety, and high patient compliance. However, many therapeutic peptides and proteins, including insulin, suffer from extremely low oral bioavailability due to multiple physiological barriers. These include enzymatic degradation by gastric and intestinal proteases, instability in acidic pH, and the limited permeability of hydrophilic macromolecules across the intestinal epithelium (Hamman et al., 2005). After oral administration, insulin is readily denatured in the stomach and degraded by proteolytic enzymes such as pepsin, trypsin, and chymotrypsin in the gastrointestinal tract (GIT), resulting in less than 1 % systemic absorption (Jawadi et al., 2022). Therefore, protecting insulin from degradation and enhancing its transepithelial transport are key challenges for successful oral delivery.

To address these limitations, numerous carrier systems have been explored, including enzyme inhibitors, permeability enhancers, and mucoadhesive polymer-based delivery systems (Khafagy et al., 2007; Andrews et al., 2009; Raeisi Estabragh et al., 2021; Zhu et al., 2016). An ideal oral peptide carrier should (i) ensure efficient protein loading and protection, (ii) maintain the biological activity of the encapsulated drug, (iii) enable sustained release at the intestinal absorption site, and (iv) be composed of biocompatible, food-grade, and non-toxic materials.
Among natural biopolymers, yeast (Saccharomyces cerevisiae) has attracted increasing attention as a safe, edible carrier. Yeast and, more specifically, the yeast cell wall (YCW) are rich in mannoproteins, β−1,3- and β−1,6-glucans, and small amounts of chitin, which confer rigidity, emulsifying capacity, and surface functionality (Kasai et al., 2000; El-Sayed et al., 2023; Yuasa et al., 2000; Yuasa et al., 2002). Yeast-based systems have shown promise in pharmaceutical applications: YCW can serve as a coating or encapsulating agent providing protection against harsh GIT conditions and enabling controlled release of encapsulated drugs (Lipke and Ovalle, 1998; Cameron et al., 1988; Wang et al., 2018). In addition, yeast components have been reported to act as absorption enhancers. In vitro studies on Caco-2 cell monolayers demonstrated that yeast extracts or cell wall fractions can transiently reduce transepithelial electrical resistance (TEER), thereby promoting paracellular permeability in a reversible and non-toxic manner (Fuller et al., 2007; Lefèvre and Subirade, 2000). The mechanism involves redistribution of tight junction proteins (e.g., ZO-1 and occludin) from the membrane to the cytoskeleton, possibly mediated by PKC activation (Fuller et al., 2007). Importantly, this effect is attributed not to intact yeast cells—which are too large to cross the paracellular space (typically 10–20 nm)—but to soluble or nanoscale YCW components such as mannoproteins and β-glucans that can interact with the epithelial surface and modulate junctional integrity.
Previous studies have highlighted the versatility of YCW in drug encapsulation. Sabu et al. (2019) developed an insulin–YCW complex coated with alginate, achieving protection against enzymatic degradation and a significant hypoglycaemic effect in diabetic rats. Similarly, YCW capsules have been used to encapsulate curcumin, improving its photostability and heat stability, while YCW-based carriers loaded with cabazitaxel or avenanthramide demonstrated sustained release and enhanced oral bioavailability (Ren et al., 2018; He et al., 2024).

In this context, the present study aimed to evaluate the effect of YCW incorporation on the physicochemical and biological properties of alginate/whey protein (ALG/WP) particles designed for oral insulin delivery. Particles were prepared by extrusion–gelation, were lyophilized and then were loaded with insulin by absorption/adsorption. After loading, beads were coated by immersion in either pure ALG solution or pure WP solution. YCW was incorporated according to two distinct strategies: (i) directly into the alginate/whey protein matrix before extrusion, or (ii) into the coating solution applied after bead formation. The formulations were characterized for insulin encapsulation efficiency, mucoadhesion, release behavior, and their effect on insulin permeability across intestinal models. We hypothesized that the inclusion of YCW could (i) improve mucoadhesion and enzymatic protection, and (ii) enhance insulin absorption by transiently modulating the paracellular pathway.

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Chemicals

Whey protein isolate (WP, Alacen® 845) was provided by NZMP (Wellington, New Zealand). Its protein content was 93 % (dry matter basis), determined by the Kjeldahl method (Nx6.38). Sodium alginate (ALG, Manucol® DH- >99 % dry matter basis) was obtained from ISP (Wayne, New Jersey, USA). Yeast cell wall (YCW) powder was obtained from LeSaffre yeast Corporation (France). Acetonitrile, methanol, calcium chloride dihydrate (CaCl2), sodium hydroxide (NaOH), hydrochloric acid (HCl), sulfate sodium anhydre (Na2SO4), phosphoric acid extra pure (H3PO4), trypsin from bovine pancreas, trifluoroacetic acid (TFA) and a human insulin ELISA kit were purchased from Fisher Scientific (Illkirch, france). Commercial human insulin solution (Umuline® rapide, 100 IU/mL) was obtained from Lilly France SAS. FITC-labeled insulin (INS-FITC, bovine pancreas) and α-chymotrypsin (bovine pancreas) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, vitamins, nonessential amino acids, l-glutamine, antibiotic solution (penicillin 103 IU/mL, streptomycin 10 mg/mL, and amphotericin B 25 μg/mL), phosphate buffered saline (PBS), trypsin-EDTA were purchased from Fisher Bioblock (Strasbourg, France).

Emmanuelle Lainé, Valerie Hoffart, Imen Dhifallah, Ghislain Garrait, Eric Beyssac, Impact of yeast cell wall incorporation on the mucoadhesion, stability, oral permeability and release profile of alginate/whey protein beads loaded with insulin, European Journal of Pharmaceutical Sciences, Volume 216, 2026, 107385, ISSN 0928-0987, https://doi.org/10.1016/j.ejps.2025.107385.

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