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

Emmanuelle Lainé, Valerie Hoffart, Imen Dhifallah, Ghislain Garrait, Eric Beyssac

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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, 2026, 216, pp.107385. 10.1016/j.ejps.2025.107385. hal-05381343

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

Emmanuelle Lainé<sup>a, *, †</sup>, Valerie Hoffart<sup>b</sup>, Imen Dhifallah<sup>a</sup>, Ghislain Garrait<sup>a</sup>, Eric Beyssac<sup>a</sup>

## ARTICLE INFO

### Keywords:

Whey protein isolate

Alginate

Yeast cell wall

Insulin

Particle

Oral administration

## 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.

## 1. 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; Lefevre

<footnote><sup>a</sup> UMR454 MEDIS, INRAE-UCA, 63000 Clermont Ferrand, France</footnote>

<footnote><sup>b</sup> UMR8258 CNRS - U1022 Inserm, UTCBS, Université Paris Descartes, Paris, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: emmanuelle.laine@uca.fr (E. Lainé).</footnote>
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.

## 2. Materials and methods

### 2.1. 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 anhydride (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).

### 2.2. Preparation of stock solutions

WP solutions (10.0 and 11.0 % w/w) were prepared by rehydrating WP isolate powder in de-ionized water under gentle magnetic stirring for 1 h at room temperature, followed by a 2 h rest period to ensure complete proteins hydration. The pH was adjusted at pH 7.0 with NaOH, and the solutions were then heated at 80 °C for 40 min to fully denature whey proteins (Lefevre and Subirade, 2000). ALG solutions (1.5 and 3.0 % w/w) were prepared in deionized water under magnetic stirring at room temperature until complete dissolution.

### 2.3. Particle preparation and insulin loading

Composite whey protein/alginate particles (WP/ALG MP, 80/20 w/w) were produced by extrusion/ionotropic gelation as described by (Hébrard et al., 2009). Briefly, the WP/ALG mixture was extruded as droplets into a 0.1 M CaCl₂ gelling bath. After gelation, the beads were collected by filtration, frozen at -80 °C, and lyophilized. Freeze-drying was performed for 48 h in a laboratory freeze-dryer. Beads were placed in the chamber (pressure < 3 Pa), maintained at -34 °C for 1 h, then progressively heated to 10 °C over 24 h. During the next 24 h, the temperature was increased and maintained at 25 °C. Lyophilized unloaded beads were loaded with insulin by absorption/adsorption. Dried beads were immersed for 5 min at room temperature in either human insulin solution or INS-FITC solution (for microscopy), then removed and gently blotted. These formulations are referred to as INS-MP (insulin-loaded particles). After loading, beads were coated by immersion in either pure ALG solution (1.5 % w/w) or pure WP solution (10.0 % w/w) for 5 min under magnetic stirring, then transferred to 0.1 M CaCl₂ for 5 min to induce film formation (Fig. 1A).

YCW were incorporated according to two strategies:

*YCW in the coating.* WP/ALG MP were coated in ALG (1.5 % w/w) or WP (10.0 % w/w) solutions containing 2 % w/w YCW, using the same immersion and CaCl₂ curing procedure (Fig. 1B),

*YCW in the matrix.* YCW (2 % w/w) was added directly to the WP/ALG solution before extrusion. The resulting YCW-containing beads (YCW-MP) were produced as above and could then be coated with pure ALG or WP (Fig. 1C).

### 2.4. Characterization of polymeric solutions (viscosity and spreadability)

The rheological behavior of polymer solutions (WP/ALG with or without insulin and/or YCW) was evaluated using a Brookfield DV-II digital viscometer (Brookfield, USA) at room temperature (n = 3 independent preparations per condition).

Spreadability was assessed (n = 3) by placing 1.00 ± 0.01 g of each formulation between two horizontal glass plates (20 × 20 cm). After 1 min under a 125 ± 1 g weight on the upper plate, the final spreading diameter was recorded following a validated protocol (Vennat et al., 1998). Increased viscosity and reduced spreadability were interpreted as indicators of improved structural integrity for bead formation.

### 2.5. Particle characterization

*Size and morphology.* Bead morphology and surface appearance were examined using a stereomicroscope (Nikon SMZ1000, Tokyo, Japan) equipped with a digital camera (Olympus Optical, Tokyo, Japan). The diameter of 90 beads per batch was measured by image analysis (Image Pro Plus). Measurements were performed on three independent batches (n = 3).

To visualize insulin distribution, INS-FITC-loaded beads were dehydrated, embedded in paraffin using an automated tissue processor (STP 120, Microm Microtech, Francheville, France), and sectioned (5 µm) at -20 °C using a microtome (HM330, Microm Microtech). Sections were observed by epifluorescence microscopy (Olympus BX-51 TF, Olympus Co. Ltd., Tokyo, Japan) with a CoolSNAP ES digital camera (Roper Scientific, Martinsried, Germany).

*Encapsulation efficiency.* Encapsulation efficiency (EE) was defined as the ratio of total entrapped insulin (quantified by RP-HPLC) to the total volume of buffer solution absorbed (determined by swelling experiment). To estimate absorbed volume and insulin loading, accurately weighed lyophilized beads [initial mass $m_0$ ≈ 130 mg; n = 3 batches] were immersed for 5 min either in insulin solution or in pH 7.0 USP buffer. Beads were removed, gently blotted, reweighed [wet mass $m(t)$], their wet diameter was measured (n = 30 beads), and they were then dried at 60 °C to constant mass [dry mass $m(dry)$]. Matrix weight loss (%), water uptake (%), and diameter change (%) were calculated using
<figure><img src="image_3.png" /><figcaption>Fig. 1. Particle preparation and coating process schema. Insulin was encapsulated by hydration of lyophilized unloaded particle by absorption/adsorption in insulin (INS-MP). (A) INS-MP were coated with pure ALG solution (1.5 % w/w) or pure WP solution (10.0 % w/w) (B) INS-MP were coated with pure ALG solution (1.5 % w/w) or pure WP solution (10.0 % w/w) containing YCW (2 % w/w), (C) YCW (2 % w/w) were added before extrusion in WP/ALG solution and particle were further coated with pure ALG solution (1.5 % w/w) or pure WP solution (10.0 % w/w).</figcaption></figure>

standard gravimetric equations:

$$ \text{Matrix weight loss (\%)} = \frac{\text{mass}(t_0) - \text{dry mass}(t)}{\text{mass}(t_0)} \times 100 $$

$$ \text{Matrix weight loss (\%)} = \frac{\text{dry mass}(t_0) - \text{dry mass}(t)}{\text{dry mass}(t_0)} \times 100 $$

$$ \text{Water uptake (\%)} = \frac{[\text{wet mass}(t) - \text{dry mass}(t)] - [\text{wet mass}(t) - \text{mass}(t_0)]}{\text{wet mass}(t) - \text{mass}(t_0)} \times 100 $$

$$ \text{Water uptake (\%)} = \frac{[\text{wet mass}(t) - \text{dry mass}(t)] - [\text{wet mass}(t_0) - \text{dry mass}(t_0)]}{\text{wet mass}(t_0) - \text{dry mass}(t_0)} \times 100 $$

$$ \text{Diameter change (\%)} = \frac{\text{wet diameter}(t) - \text{wet diameter}(t_0)}{\text{wet diameter}(t_0)} \times 100 $$

$$ \text{Diameter change (\%)} = \frac{\text{wet diameter}(t) - \text{wet diameter}(t_0)}{\text{wet diameter}(t_0)} \times 100 $$

Insulin content was quantified by reverse-phase HPLC as described below (Section 2.6).

Swelling studies. The swelling behaviors of MP containing or not INS and YCW was evaluated after 15 min of dissolution: MP (2.4 g) were introduced in 50 mL buffer pH 1.2 or pH 6.8 at 37 °C in Schott Duran® flask (50 rpm). After 15 min, all MP were withdrawn (in triplicate), accurately weighed [wet mass (t)], measured as described above [wet diameter (t)] and dried to constant mass at 60 °C [dry mass (t)]. The matrix weight loss ( %), water uptake ( %) and diameter changes ( %) were calculated as follows:

## 2.6. Functional contribution of YCW

In vitro insulin release. Insulin releases from uncoated and coated INS-MP containing or not YCW in the matrix or in the coating, were investigated in 50 mL buffer pH 1.2 or pH 6.8 at 37 °C in Schott Duran® flask (50 rpm) during 4 h. Samples withdrawn (1 mL) at predetermined times, were centrifuged (5000 rpm, 5 min) and insulin quantities were measured using a RP-HPLC method described above. Briefly, 20 µL of the samples were injected via an autosampler (AS-2000 A) connected to an intelligent pump (L-62,000 A), a diode array detector (L-45,000) and
an interface (D-6000). The LiChrospher RP-18 column (5 µm, 4 × 250 mm, Merck, Darmstadt, Germany) was equilibrated at 40 °C, with column thermostat (L-5025), at a flow rate of 0.8 mL/min with 72 % eluent A (56.8 g Na₂SO₄, 2 L ultra-pure water and pH adjusted at 2.3 with H₃PO₄) and 28 % eluent B (acetonitrile). The absorbance of the eluent was recorded at 214 nm. The column was calibrated with insulin solutions of known concentrations. This HPLC method was consistently used for release, enzymatic degradation, and permeability studies. ELISA was used only to confirm that insulin retained its immunoreactivity. All experiments were performed in triplicate. The insulin release data were expressed as the cumulative percentage of insulin released versus time. For quantitative comparison, the area under the release curve from 0 to 4 h (AUC₀–4 h) was calculated for each replicate using the trapezoidal rule:

$$AUC = \sum_{i=1}^{n-1} \frac{(t_{i+1} - t_i)(R_{i+1} + R_i)}{2}$$

where $t_i$ and $R_i$ are the sampling time and the percentage of insulin released at each interval, respectively. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test using GraphPad Prism 9. Differences were considered significant for $p < 0.05$.

To analyze the release mechanism, the data were also fitted to the semi-empirical model of (Harland et al., 1988).

$$Mt/M\infty = A\sqrt{t} + Bt$$

In the above equations, $Mt/M\infty$ is the fraction of drug released at time t, A and B are diffusion and erosion terms. When $A > B$, the diffusion factor prevails in the release system, while when $A < B$, erosion predominates. If $A = B$, the release mechanism includes both diffusion and erosion equally.

**Enzyme inhibitory effect.** The capacity of the formulations to inhibit intestinal proteases was evaluated following (Sajeesh et al., 2010; Khafagy et al., 2007).

Trypsin and α-chymotrypsin were selected for the enzymatic degradation assay because they represent the two major serine proteases responsible for protein and peptide hydrolysis in the small intestine. After gastric emptying, pancreatic secretions provide a high concentration of these enzymes (0.1–1 mg/mL), which rapidly cleave peptide drugs such as insulin at specific residues. Trypsin preferentially hydrolyses at the C-terminal side of lysine and arginine, while α-chymotrypsin cleaves aromatic residues (phenylalanine, tyrosine, tryptophan). Together, they account for the dominant proteolytic activity acting on orally administered insulin in the duodenum and upper jejunum. Previous studies have demonstrated that insulin is rapidly degraded in the presence of these enzymes, with >80–90 % loss of intact peptide within minutes, making them the most relevant in vitro model for predicting intestinal proteolysis. Including both enzymes therefore allow a representative simulation of the proteolytic barriers encountered by insulin upon oral administration and provides a meaningful assessment of the protective or inhibitory capacity of YCW-containing formulations.

- Trypsin inhibition. Beads were dispersed in phosphate buffer (0.2 M, pH 7.4) to obtain a final 1 % (w/v) suspension. One milliliter of this dispersion was mixed with 1 mL of BAPNA substrate (20 mM in DMF) and 30 units of trypsin (prepared in 10 mM HCl), then incubated for 30 min at 37 °C. The reaction was stopped with 1 % trichloroacetic acid, and the amount of p-nitroaniline formed was quantified at 405 nm. Control reactions were performed identically but without beads. The activity of non-inhibited trypsin was defined as 100 %, and residual activity after incubation with beads was expressed as % of this value.

- α-Chymotrypsin inhibition. A similar protocol was used with BTPNA (1.18 mM in DMF) as substrate in Tris–HCl buffer (pH 7.8). Bead dispersions (1 % w/v) were incubated with 40 units of

α-chymotrypsin for 30 min at 37 °C. After quenching with 1 % trichloroacetic acid, inhibition (%) was calculated as described above for trypsin.

**Insulin protection against enzymatic degradation.** To assess protection against enzymatic degradation, INS-MP and INS-YCW-MP were dispersed in USP pH 7.4 buffer containing trypsin (10 IU/mL) and α-chymotrypsin (10 IU/mL) and incubated at 37 °C. At predetermined time points, aliquots were withdrawn and the reaction was stopped with 0.1 % TFA in water. The insulin present in the medium was quantified by RP-HPLC, and the remaining insulin associated with the beads was quantified after transferring the beads to fresh buffer and extracting. The biological activity of the released insulin was confirmed by ELISA. Controls included native insulin incubated with enzymes in the absence of beads, and samples incubated without enzymes to determine the % of intact (undegraded) insulin. All experiments were performed in triplicate ($n = 3$ independent batches).

**Mucoadhesive.** Mucoadhesion studies were done on rabbit duodenum mucosa as described by (Rao and Buri, 1989) with different particles: glass beads (1000–1200 µm diameter), MP and YCW-MP. Experiments also included YCW powder for additional assessment. Glass beads served as a control due to their comparable surface area with the polymeric MP. Briefly, intestinal tissue from duodenum (10 cm length) was taken, flushed with physiological serum (0.9 % NaCl) to remove free mucus on mucosal side and cut open longitudinally. The tissue was mounted on a plastic support at an angle of 45° and washed with physiological serum. Twenty particles or 200 µg of powder were applied to the mucosal surface and kept 5 min for particle- or powder-mucosal interactions. The tissue was then washed with pH 6.8 USP buffer for 30 min (flow rate 24 mL/min). The number of particles still attached on the duodenal tissue after 5, 10 or 30 min washing time was counted manually. For YCW powder, which cannot be individually counted, the adhered material was collected with mucus, rinsed, and weighed after drying. Mucoadhesive capacity was expressed as the percentage of particles or powder retained on the mucosal tissue at the end of the process. Experiment was performed in triplicate.

## 2.7. Effect of YCW on insulin permeability in Caco-2 cells

**Cell culture.** The Caco-2 human colon cancer cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) at passage 35–40. Stock cultures were maintained in DMEM containing 4.5 g/L of glucose supplemented with 20 % fetal bovine serum, 2 % vitamins, 2 % nonessential amino acids, 2 % L-glutamine, and 2 % of an antibiotic solution, at 37 °C in a humidified atmosphere of air/CO₂ (95:5, v/v). The medium was changed every 3 days. Plates of culture 6 wells Transwell polycarbonate cell culture supports were seeded with $5 \times 10^5$ cells per well, and the medium was refreshed daily. All experiments were carried out 21 days after initial seeding when Caco-2 cells exhibited maximum differentiation. Before each experiment, the medium was removed, and the cell monolayer was washed three times with 1 mL of PBS. Several treatments were tested on apical Caco-2 monolayer. When Transwell cell culture supports were used, the transepithelial electrical resistance (TEER) was also measured (electrode STX2, EVOMX World Precision Instruments, Sarasota, FL, USA).

**Monolayer integrity and cell viability.** Cytotoxicity studies were performed to evaluate the viability of cells in the presence of INS, YCW or MP. One milliliter of free INS solution (F-INS, 300 µg/mL) and free INS-YCW solution (F-INS-YCW) were tested. As for F-INS tested on Caco-2 cells, INS-MP (corresponding to insulin of 300 µg/mL) and INS-YCW-MP (corresponding to insulin of 300 µg/mL) were suspended in 1 mL of DMEM and applied to the apical compartment. Cells were incubated at 37 °C during 120 min and the viability was evaluated by trypan blue exclusion (final concentration of 0.1 %). TEER values of the monolayers were also measured to verify the monolayers confluence and integrity.

**Effect of YCW on TEER.** Measurements were conducted before
treatments and at predetermined times with control: no treatment (NT), unloaded MP (MP) and experiments: free-YCW (F-YCW) and YCW loaded MP (YCW-MP). After 120 min, the treatment medium (solution and/or MP) was removed and cells were washed with PBS, fresh medium was immediately replaced, and cells were incubated at 37 °C. The monolayers were allowed to regenerate for 48 h at 37 °C in an atmosphere of 95 % air and 5 % CO2 at 90 % relative humidity. As a measure of cell viability and monolayer integrity, TEER was recorded throughout incubation and during 48 h for recovery period.

The TEER was calculated according to Eq. (1).

$$TEER(\Omega.cm^2) = (R_{\text{total}} - R_{\text{filter}}) \times A \quad (1)$$

where $R_{total}$ ($\Omega$) is the measured resistance, $R_{filter}$ ($\Omega$) is the resistance of the filter without cells, and A is the surface area (cm$^2$).

**Insulin permeability.** To assess transport, Free INS solution (F-INS, 300 µg/mL), free INS-YCW solution (F-INS-YCW, equivalent to 300 µg/mL), INS-MP (equivalent to 300 µg/mL) and INS-YCW-MP (equivalent to 300 µg/mL) were suspended in 1 mL of DMEM and added to apical compartment. Cells were incubated at 37 °C during 120 min. After 120 min, 50 µL of solution in basal compartment were taken out and the INS concentration was determined by RP-HPLC analysis. All experiments were performed in triplicate. Results are expressed as apparent permeability coefficient ($P_{app}$), calculated according to Eq. (2).

$$P_{\text{app}}(\text{cm.s}) = (dQ/dt) \times [1/(A \cdot C_0)] \quad (2)$$

where $dQ/dt$ is the flux of insulin across the monolayer (mol/sec), $C_0$ is the initial concentration of insulin in the apical compartment (mol/mL), and A is the surface area of the monolayer (3.14 cm$^2$). The $P_{app}$ values are expressed as cm.sec$^{-1}$. The $P_{app}$ values were calculated for the 120 min period of the experiment.

Recovery rates were calculated as controls of compound adsorption to cells and/or support, according to Eq. (3), to verify the mass balance of each experiment:

$$\% \text{Recovery} = (Q_{\text{basolateral}}_{t120} + Q_{\text{apical}}_{t120}) / Q_{\text{apical}}_{t0} \quad (3)$$

where $Q_{basolateral}$ t120 is the total amount of insulin in the basolateral compartment after 120 min incubation, $Q_{apical}$ t120 is the total amount of insulin remaining in the apical compartment, and $Q_{apical}$ t0 is the total amount of insulin initially added to the apical compartment. All Caco-2 experiments were performed in triplicate (n = 3 inserts per condition).

## 2.8. Ex vivo permeability in excised intestinal tissue

**Animals.** Animal protocols were approved by the Committee for Ethical Issues, CEMEA Auvergne (Apafis, 18,653–2019012515 397076V3, Clermont-Ferrand, France). Adult male Wistar rats weighing 300 ± 20 g, were housed under controlled conditions, with a temperature-of 22 ± 3 °C and a 12 h light/dark cycle (lights on from 8:00 a.m. to 8:00 pm.). Animals had free access to a commercial pelleted standard and tap water. After fasting for 12–16 h, rats were anesthetized using Imalgene 1000® and Valium 10® (150 µL and 50 µL/100 g of rat respectively) and locally with 1 % Xylocaine®.

**Intestinal sac assay.** Rats were divided into 3 groups of 5 animals. A segment of 10 cm from the duodenum of each rat was quickly removed and rinsed with 10 mL of physiological serum (0.9 % NaCl) maintained at 37 °C. One end of the segment was ligated, INS-YCW-MP (corresponding to insulin concentration of 300 µg/mL) were introduced on the intestinal lumen and then the second end was ligated. Control experiments were performed with free insulin solution (300 µg/mL) and INS-MP (300 µg/mL). Gastrointestinal sacs were immediately transferred into a tissue chamber containing 15 mL of warmed (37 °C), oxygenated (95 % O$_2$/5 % CO$_2$) Krebs Henseleit modified buffer (composition in mmol/L: 118.1 NaCl, 4.7 KCl, 2.2 CaCl$_2$·2H$_2$O, 1.2 MgSO$_4$, 1.2 KH$_2$PO$_4$,

25.0 NaHCO$_3$, and 11.1 glucose, pH 7.5). Samples were collected (0.5 mL) from the serosal side after 120 min. Insulin content was measured by RP-HPLC and immunoreactivity was confirmed by ELISA to ensure detection of intact insulin. The apparent permeability coefficient ($P_{app}$) was calculated according to Eq. (4):

$$P_{\text{app}} = \left( \frac{dQ}{dt} \right) \times \left( \frac{1}{A C_0} \right) \quad (4)$$

with $dQ/dt$, the flux of INS from the mucosal to the serosal side of the mucosa; $C_0$, the initial concentration of insulin in the lumen compartment and A, the membrane area ($A$ = length x diameter). The absorption enhancement ratio was calculated according to Eq. (5):

$$R = \frac{P_{\text{app}}(\text{sample})}{P_{\text{app}}(\text{control})} \quad (5)$$

## 2.9. In vivo intestinal absorption and pharmacokinetics in rats

**Animals.** Animal protocols were approved by the Committee for Ethical Issues, CEMEA Auvergne (APAFIS #29,153–2021,011, 417,345,249 v2, Clermont-Ferrand, France).

**Duodenal administration model.** Pharmacokinetic studies were carried out in healthy male Wistar rats weighing as described before. Rats were divided into 4 groups of 6 animals. Because the particles are approximately 1.4 mm in diameter and cannot be reliably administered by gavage, we used a direct duodenal administration model previously described for intestinal absorption studies (Whitehead et al., 2004). Under anesthesia, a midline abdominal incision (~1.5 cm) was made to expose the proximal small intestine. A small longitudinal incision (0.5 cm) was made about 2 cm after the stomach. INS-YCW-MP (corresponding to human insulin concentration of 730 µg/mL) were inserted through the opening into the lumen. Negative control experiments were performed by the insertion of free INS solution (human insulin of 730 µg/mL) and INS-MP (human insulin of 730 µg/mL) in the intestinal lumen in the same way. The incisions were then sealed with suture. Positive controls were performed by subcutaneously injection of human insulin solution (63 µg/mL). Before experiment and after 120 min, blood samples (300 µL) were collected from catheterized carotid vein, into heparinized tubes. Blood was centrifuged at 10,000 g for 5 min, and plasma was separated and stored at –20 °C until further analysis. Immunoreactivity of plasmatic human insulin was assessed using ELISA technique as per the standard protocol. Human insulin ELISA kit does not cross-react with rat insulin. Therefore, endogenous insulin did not interfere with the results, thus allowing selective detection of administered human insulin. The pharmacological availability (PA) of the orally administered insulin was calculated as follows (6):

$$PA (\%) = \left( \frac{AUC_{\text{duodenal}}}{\text{dose}_{\text{duodenal}}} \times 100 \right) \div \left( \frac{AUC_{\text{sc}}}{\text{dose}_{\text{sc}}} \right) \quad (6)$$

where $AUC_{duodenal}$ is the area determined after duodenal administration of INS-YCW-MP, INS-MP and F-INS and $AUC_{sc}$ is the area determined after subcutaneous injection of insulin solution.

**Confocal imaging of intestinal uptake.** To visualize local delivery, rats were divided into 2 groups (2 animals per group): unloaded MP (control), INS-FITC loaded YCW-MP (INS-FITC-YCW-MP). Samples were introduced in duodenum of rat as described in the *in vivo* evaluation. Duodenal tissue not exposed to fluorescent-labelled MP (unloaded MP) was used as control to exclude the possibility of autofluorescence associated with tissues. After a 60 min incubation period, a segment of 2 cm from the experimented duodenum was quickly ligated at both end and then removed. Snap frozen duodenum in isopentane were embedded into optimal cutting temperature medium (OCT) and stored at –80 °C. Seven micrometres of frozen duodenum were cut in a cryostat. Then Duodenum mucosa were fixed in 1 % PFA for 20 min, washed in phosphate buffer saline (PBS) and nucleus were stained with Hoechst stain.
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Influence of INS and YCW on the viscosity and the diameter spread of WP/ALG solution (80/20 WP/ALG). The results are presented as mean ± standard deviation (n = 3). Viscosity of INS-WP/ALG (INS), YCW-WP/ALG (YCW) and INS-YCW-WP/ALG (INS-YCW) solutions statistically different from the control (no treatment NT: WP/ALG): ***p < 0.001.</figcaption></figure>

Slides were mounted using Mountex-mounting medium (CellPath). Tissues were visualized using a confocal microscope Zeiss LSM 510 Meta (Carl Zeiss, Inc). Non-exposed duodenum was used as a negative control to exclude tissue autofluorescence.

## 2.10. Statistical analysis

All experiments were performed at least in triplicate unless otherwise stated. For *in vitro* and *ex vivo* assays, *n* = 3 independent preparations per condition were used. For animal studies, *n* = 5–6 animals per group. Data are expressed as mean ± standard deviation (SD). One-way ANOVA followed by Tukey's post hoc test (GraphPad Prism 9) was used for group comparisons. A value of *p* < 0.05 was considered statistically significant.

# 3. Results and discussion

The formulation concept of insulin-loaded whey protein/alginate (WP/ALG) beads containing yeast cell wall (YCW) was developed to increase insulin bioavailability in the gastrointestinal tract by (i) protecting insulin against acidic pH and proteolytic degradation, (ii) promoting mucoadhesion at the intestinal surface, and (iii) transiently enhancing paracellular transport to improve intestinal permeability.

## 3.1. Influence of YCW on polymeric solution characteristics

The viscosity and spreadability of the polymeric solutions prepared with different formulations are shown in Fig. 2. A strong inverse correlation (R² = 0.9903) was observed between viscosity and spreading diameter: higher-viscosity solutions produced smaller spread diameters. The addition of insulin alone significantly decreased solution viscosity and increased spreadability, indicating that insulin interfered with protein–polysaccharide interactions and generated a less structured gel network. Such a decrease in viscosity is unfavorable for the production of mechanically stable and spherical beads.

Importantly, incorporation of YCW restored the viscosity of the solutions, counteracting the thinning effect of insulin and enabling the formation of spherical, mechanically robust beads. This suggests that YCW contributes structurally to the formulation by increasing the density of intermolecular interactions in the precursor hydrogel. Matrix/

<caption>**Table 1**</caption>

Influence of YCW added in the particle (MP) or in the coating, on the encapsulation efficiency, the INS loading and the mean size of MP. The results are presented as mean ± standard deviation (n = 3).

<table><thead><tr><th>Formulation</th><th>Coating</th><th>Encapsulation efficiency (%)</th><th>Insulin loading (IU/75 MP)</th><th>Mean size (μm)</th></tr></thead><tbody><tr><td rowspan="3">Control</td><td>MP</td><td>-</td><td>98.0 ± 2.0</td><td>31.4 ± 0.6</td><td>1467 ± 101</td></tr><tr><td>MP</td><td>WP</td><td>57.2 ± 7.3<sup>a,b</sup></td><td>18.4 ± 0.8<sup>a,b</sup></td><td>1535 ± 92</td></tr><tr><td>MP</td><td>ALG</td><td>60.7 ± 2.0<sup>a,b</sup></td><td>19.4 ± 0.6<sup>a,b</sup></td><td>1562 ± 91<sup>a</sup></td></tr><tr><td rowspan="6">Experiments</td><td>MP</td><td>WP-YCW</td><td>65.1 ± 1.3<sup>a,b</sup></td><td>20.9 ± 0.4<sup>a,b</sup></td><td>1538 ± 98</td></tr><tr><td>MP</td><td>ALG-YCW</td><td>71.4 ± 1.3<sup>a,b</sup></td><td>22.8 ± 0.4<sup>a,b</sup></td><td>1570 ± 94<sup>a</sup></td></tr><tr><td>YCW- MP</td><td>-</td><td>98.7 ± 0.8</td><td>31.5 ± 0.2</td><td>1470 ± 90</td></tr><tr><td>YCW- MP</td><td>WP</td><td>83.1 ± 1.1<sup>a,b,c</sup></td><td>26.7 ± 0.4<sup>a,b,c</sup></td><td>1539 ± 86</td></tr><tr><td>MP</td><td>ALG</td><td>84.1 ± 2.6<sup>a,b,d</sup></td><td>26.9 ± 0.8<sup>a,b,d</sup></td><td>1569 ± 90<sup>a,b</sup></td></tr></tbody></table>

Mean size:.

a statistically different from MP, *p* < 0.01.

b statistically different from YCW-MP, *p* < 0.01
Encapsulation efficiency and insulin loading:.

c statistically different from MP, *p* < 0.001.

d statistically different from MP, *p* < 0.001.

e statistically different from MP coated WP-YCW, *p* < 0.0001.

f statistically different from MP coated ALG-YCW, *p* < 0.01.

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Photographs of (A) INS-particle and (B) INS-YCW-particle before lyophilisation (scale bar represents 1 mm) and distribution of insulin-FITC on the inside (cross section) of particle.</figcaption></figure>
insulin ionic interactions occurring during bead formation (Whitehead et al., 2004; Pamies et al., 2010) can alter gelation behavior, and small changes in WP concentration strongly affect viscosity, sometimes yielding deformed or fragile beads. The presence of YCW stabilized the precursor mixture, which likely helped to retain insulin within the bead during subsequent processing (loading and coating). These findings agree with previous reports that yeast or YCW incorporation can increase viscosity and improve mechanical stability of biopolymer solutions (Yuasa et al., 2002; Zhao et al., 2009; Hébrard et al., 2010).

## 3.2. Influence of YCW on particle characteristics

**Beads preparation.** Insulin-loaded particles (INS-MP) were produced by extrusion and cold Ca²⁺-induced gelation of WP/ALG hydrogels with or without YCW (Fig. 3). After gelation, beads were lyophilized and then loaded with insulin by absorption/adsorption from an insulin solution. Microscopy confirmed that the beads remained spherical after freezing-drying, indicating that the drying step did not collapse the structure (Fig. 3). The mean bead diameter was approximately 1467 ± 101 µm and did not differ significantly from the YCW-containing uncoated beads (YCW-MP = 1470 ± 90 µm) (Table 1). Incorporation of YCW at 2 % w/w therefore did not alter the extrusion droplet formation or the gelation process, resulting in similar bead size distribution.

Encapsulation efficiencies (EE) were generally high, though markedly affected by the coating type (Table 1). The uncoated control MP displayed an EE of 98.0 ± 2.0 %, whereas coating with WP or ALG significantly reduced EE to 57.2 ± 7.3 % and 60.7 ± 2.0 %, respectively (*p* < 0.001 vs uncoated MP). This drop likely results from partial insulin leaching during the wet coating step. When YCW was incorporated either into the coating (WP-YCW or ALG-YCW) or directly into the bead matrix (YCW-MP), EE significantly increased compared with the corresponding controls (*p* < 0.001). The highest EEs were obtained for YCW-MP (98.7 ± 0.8 %) and for coated YCW-MP formulations (83.1 ± 1.1 % for WP; 84.1 ± 1.2 % for ALG), demonstrating that YCW effectively limited insulin diffusion during coating and drying. Insulin loading followed a similar pattern, ranging from 18.4 ± 0.8 to 31.5 ± 0.2 IU/75 MP. The highest loading was again obtained with YCW-MP (31.5 ± 0.2 IU/75 MP), significantly greater than that of all coated formulations (*p* < 0.001). Among the coated beads, ALG-YCW and WP-YCW coatings resulted in higher insulin content than their YCW-free counterparts (*p* < 0.01). This improvement can be attributed to the viscosity-enhancing effect of YCW, which stabilizes the polymeric matrix and prevents insulin diffusion during coating.

Confocal imaging of FITC-labeled insulin (INS-FITC) confirmed a homogeneous distribution of insulin within the bead structure and localized fluorescence associated with YCW domains, supporting the physical entrapment and possible electrostatic association between insulin and YCW polysaccharides (Fig. 3). The average bead diameter (~1.5 mm) is larger than the strict <1000 µm definition often used for “microparticles.” For accuracy, we now refer to these systems as “beads or particles”. This is also why, *in vivo*, direct oral gavage was not used; instead, beads were placed directly into the duodenum (Section 2.6), which isolates the intestinal absorption step.

**Coating.** INS-MP and INS-YCW-MP were successfully coated with ALG, WP, ALG+YCW, or WP+YCW by a simple immersion and Ca²⁺ curing procedure, yielding individually coated spherical beads. Coating thickness was evaluated by comparing diameters of coated vs uncoated beads (Table 1).

All coated formulations displayed slightly higher mean diameters (ranging from 1535 ± 92 µm to 1570 ± 94 µm) compared with the uncoated control, confirming the successful deposition of an external layer. Statistically significant increases in size (*p* < 0.01) were observed for ALG-coated formulations, ALG-YCW-coated formulation compared with uncoated MP, whereas no difference was found between coatings containing or not containing WP and YCW, suggesting that YCW did not influence coating thickness.

<figure><img src="image_6.png" /></figure>

**Fig. 4.** Evaluation of the matrix weight loss, water uptake and diameter change of controls: unloaded particle (MP), INS loaded particle (INS-MP) and treatments: unloaded YCW-particle (YCW-MP), INS-YCW loaded particle (INS-YCW-MP), after dissolution for 15 min, at (A) gastric pH (1.2) or (B) intestinal pH (6.8). The swelling was evaluated by size and weight measurements of particle for the diameter change, the water uptake and the matrix weight loss. The results are presented as mean ± standard deviation (n = 90 for diameter change and n = 6 for water uptake and matrix weight loss). YCW-MP statistically different from MP: \*p* < 0.05; \*\*\*p* < 0.001 and INS-YCW-MP statistically different from INS-MP: \*\*\*\*p* < 0.001.

Although YCW is often handled as an insoluble particulate/suspension rather than a true polymer solution (Kasai et al., 2000), its inclusion in the coating process reduced insulin loss during coating. Insulin leakage during coating decreased by ~10 % (ALG) and ~8 % (WP) when YCW was added to the coating solution, and by ~22–25 % when YCW was incorporated into the bead core before coating. We attribute this improved retention to the increased viscosity and structural cohesion provided by YCW, which likely slows insulin diffusion out of the beads during the wet coating step and stabilizes insulin-matrix interactions.

**Particle swelling.** Swelling behavior (matrix weight loss, water uptake, and diameter change) at gastric pH 1.2 and intestinal pH 6.8 is presented in Figs. 4 and 5. At both pH values, insulin loading significantly increased water uptake, diameter expansion, and matrix weight loss compared to unloaded beads (Fig. 5A). This is consistent with electrostatic repulsion between negatively charged ALG chains and insulin, which destabilizes the network and promotes water penetration. At pH 6.8, ALG carboxylate groups are deprotonated, which increases electrostatic repulsion and promotes polymer relaxation and swelling; this explains the higher swelling and higher mass loss observed under intestinal pH compared to acidic pH.

Incorporation of YCW altered the swelling profile. YCW-containing beads showed greater matrix relaxation and higher structural weakening (Fig. 5B) than beads without YCW (Fig. 5A), suggesting that YCW disrupts polymer-polymer packing, possibly by interfering with hydrogen bonding and other weak intermolecular forces (e.g. van der Waals interactions) between WP and ALG. Similar modulation of network cohesion by yeast-derived polysaccharides has been described in protein/polysaccharide systems and in YCW-containing coatings (Yuasa et al., 2002; Hébrard et al., 2010). These data indicate that while YCW contributes beneficially to viscosity and insulin retention during fabrication, it may also promote structural softening and susceptibility to deformation during swelling, particularly at intestinal pH where the gel is already more relaxed.

Overall, swelling behavior is important because it anticipates release
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Schematic representation of YCW impact on particle swelling. (A) Solution of ALG and WP polymers were extruded in calcium bath and INS was encapsulated in particle by absorption/adsorption, (B) YCW were incorporated in particle before extrusion/gelation in calcium bath and INS was then encapsulated.</figcaption></figure>

kinetics. Formulations that absorb more water and undergo faster structural relaxation at pH 6.8 are expected to release insulin more rapidly by diffusion.

### 3.3. Functional contribution of YCW to insulin delivery

*In vitro* insulin release. To overcome gastrointestinal barriers, an ideal oral formulation should (i) limit insulin release in the stomach and (ii) ensure rapid availability at the intestinal absorption site. We therefore evaluated insulin release from all bead formulations in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 6.8) over 4 h (Fig. 6).

In both media, the release profiles from uncoated YCW-MP (Fig. 6A), and from coated MP using ALG/WP (Fig. 6B) or ALG-YCW/WP-YCW polymers (Fig. 6C), exhibited an initial burst followed by a slower release phase. Insulin release was clearly governed by a diffusion mechanism, as demonstrated by the Harland model ($A > B$, Table 2), rather than by erosion. The initial burst can be attributed to the diffusion of insulin molecules located near the bead surface, whereas the gradual phase likely corresponds to diffusion from the inner polymeric matrix. These observations are in agreement with the swelling experiments described previously, which showed limited matrix erosion under both pH conditions.

To quantitatively compare release kinetics, the area under the release curve (AUC~0~4 h) was calculated for each replicate using the trapezoidal rule, followed by statistical analysis ($n = 3$). The results (Fig. 6, right panels) showed that insulin release was significantly faster at pH 6.8 than at pH 1.2, as reflected by higher AUC values ($**p < 0.001$), consistent with the pH-dependent relaxation of alginate networks. At acidic pH, alginate remains compact due to protonation of carboxyl groups, while at pH 6.8 deprotonation increases electrostatic repulsion, leading to greater swelling and enhanced diffusion. When comparing uncoated formulations, the incorporation of yeast cell wall (YCW-MP) produced only a modest increase in AUC~0~4 h at pH 1.2 ($**p < 0.001$) and no significant difference at pH 6.8 (ns, $p > 0.05$). This indicates that YCW did not markedly affect insulin diffusion in neutral medium, although it may slightly weaken the polymeric network under acidic conditions by reducing chain-chain interactions within the ALG/WP matrix. In coated systems, all formulations (WP-coated, ALG-coated, WP-YCW-coated, and ALG-YCW-coated MP) displayed rapid insulin release, reaching 80–100% within the first hour. Statistical comparison confirmed significant differences between uncoated and coated formulations ($**p < 0.0001$) but no meaningful variation among coatings of different composition, showing that neither the type of polymer nor the

incorporation of YCW in the coating significantly altered AUC~0~4 h values. The absence of prolonged release from coated beads is explained by the nature of the coatings used: thin, food-grade protein or alginate layers formed by immersion, which become rapidly hydrated and permeable in aqueous media. These coatings were efficient in limiting insulin loss during bead preparation but did not behave as enteric or diffusion-controlling barriers during dissolution. Consequently, insulin release remained mainly governed by diffusion through a hydrated polymeric network rather than by erosion or coating resistance.

Our results were not in accordance with those already published by (Yuasa et al., 2000; Yuasa et al., 2002) with acetaminophen tablets coated with acid-treated YCW aqueous dispersion containing 5% (w:v) of acid-treated YCW and 0.35% (w:v) of glycerol. Acetaminophen release from the coated tablets showed a sigmoidal release profile with an initial lag time. However, Yuasa et al. (2002) has also tested the coating for granules. Unlike tablets, the release from coated granules was obviously rapid as in our results.

Despite the absence of a strong release-retarding effect, the mucoadhesive and protective properties of YCW and the polymeric matrix remain advantageous. The adhesive nature of these formulations may promote a localized microenvironment at the intestinal surface, favoring drug absorption. Nevertheless, for oral administration, an additional gastroresistant barrier should be considered-either by applying a thicker coating in a fluidized bed system or by incorporating the particles into enteric-coated capsules, as suggested by Ivanova, 2021. Under these conditions, a rapid drug release in the intestinal tract would be expected. Similarly, studies on probiotic encapsulation demonstrated that *Lactobacillus acidophilus* coated with S. cerevisiae cell wall exhibited improved resistance to gastric conditions, further supporting the protective effect of yeast-derived coatings (Utama et al., 2023).

**Enzyme inhibitory effect of YCW.** Rapid release alone is not sufficient for oral delivery if insulin is immediately degraded by intestinal proteases. YCW encapsulation could still contribute to increase oral bioavailability of insulin thanks to their potential enzyme inhibitory properties. Thus, a rapid release close to intestinal border can still favored drug absorption if insulin can be protected towards enzymatic degradation. This approach is particularly valuable because some polymers had shown already enzyme inhibitory effect (Sajeesh et al., 2010), which can aid in preserving protein during oral delivery. In this work, YCW inhibitory effect was evaluated. Trypsin and α-chymotrypsin were used as model proteases to study the inhibition process because they play a vital role in denaturating orally administrated proteins (Sajeesh and Sharma, 2011). *In vitro* trypsin and α-Chymotrypsin
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** *In vitro* release profiles of insulin from alginate/whey protein particles with or without yeast cell wall (YCW). (A) Uncoated particle: MP (♦) and YCW-MP (▲). (B) Coated particle: WP coated MP (◆, solid line), ALG coated MP (◆, dotted line), WP coated YCW-MP (▲, solid line), ALG coated YCW-MP (▲, dotted line). (C) Coated particle prepared using polymer blends containing or not YCW: WP coated MP (◆, solid line), ALG coated MP (◆, dotted line), WP-YCW coated MP (▲, solid line), ALG-YCW coated MP (▲, dotted line). Experiments were performed in simulated gastric fluid (hydrochloric acid, pH 1.2, black symbols) and simulated intestinal fluid (phosphate buffer, pH 6.8, white symbols) at 37 °C for 4 h. Data are presented as mean ± SD (n = 3). To quantitatively compare release kinetics, the area under the release curve (AUC<sub>0–4h</sub>) was calculated for each replicate using the trapezoidal rule and statistical analysis (one-way ANOVA with Tukey's post-hoc test, n = 3) were performed.</figcaption></figure>

inhibitory effect of polymers and YCW is given in the Fig. 7. YCW-MP permit a 65 and 70 % inhibitory effect for trypsin and α-Chymotrypsin activity respectively, and this inhibition was significantly superior to WP/ALG MP. The action of enzymes on substrate was decreased, suggesting that the enzyme preferentially breaks down a certain portion of peptides of YCW and then its proteolytic activity is repressed. This suggests that the enzymes preferentially interact with and partially digest YCW-derived peptides or polysaccharide-associated protein domains, thereby reducing their effective activity toward insulin. Such “sacrificial substrate” behavior has been suggested for polysaccharide- and protein-based enzyme inhibitors (Sajeesh et al., 2010).

**Protection of insulin against enzymatic degradation.** Insulin degradation was next evaluated in the presence of trypsin and chymotrypsin at pH 7.4 over 120 min (Fig. 8). After 60 min, approximately 60 % of insulin

remained intact when encapsulated in either MP or YCW-MP, whereas free insulin alone was rapidly degraded. ELISA confirmed that the insulin remaining associated with the beads retained immunoreactivity, indicating preservation of structural integrity.

Interestingly, despite the stronger enzyme inhibition seen with YCW-MP, there was no significant difference between MP and YCW-MP in terms of the fraction of insulin protected over 60 min. This is likely because, at intestinal pH, more than 90 % of the insulin is released within the first hour for all formulations; thus, once insulin diffuses into the medium, both formulations become similarly exposed to enzymes. In other words, YCW helps inhibit enzymes, but the very fast release limits the window during which that advantage can be expressed.

Overall, these data indicate that protection against enzymatic degradation is provided primarily by transient physical association of
<caption>**Table 2**</caption>

Influence of YCW added in particle (MP) or in coating on INS release mechanism (diffusion or erosion) in pH 1.2 and pH 6.8 buffers, fitted to equation proposed by Harland, compared to the control (MP).

<table><thead><tr><th rowspan="2">Dissolution medium</th><th rowspan="2">Formulation</th><th colspan="2">Harland equation</th><th rowspan="2">Correlation coefficient</th><th rowspan="2">Mechanisms release</th></tr><tr><th>A</th><th>B</th><th>r</th></tr></thead><tbody><tr><td rowspan="10">pH 1.2 buffer</td><td>MP</td><td>0.577</td><td>0</td><td>0.987</td><td>Diffusion</td></tr><tr><td>YCW-MP</td><td>0.508</td><td>0</td><td>0.931</td><td>Diffusion</td></tr><tr><td>WP coated MP</td><td>0.575</td><td>0</td><td>0.955</td><td>Diffusion</td></tr><tr><td>ALG coated MP</td><td>0.540</td><td>0</td><td>0.970</td><td>Diffusion</td></tr><tr><td>WP coated YCW-MP</td><td>0.488</td><td>0</td><td>0.926</td><td>Diffusion</td></tr><tr><td>ALG coated YCW-MP</td><td>0.474</td><td>0</td><td>0.959</td><td>Diffusion</td></tr><tr><td>WP-YCW coated MP</td><td>0.519</td><td>0</td><td>0.852</td><td>Diffusion</td></tr><tr><td>ALG-YCW coated MP</td><td>0.556</td><td>0</td><td>0.895</td><td>Diffusion</td></tr><tr><td>WP-YCW MP</td><td>0.901</td><td>0</td><td>0.965</td><td>Diffusion</td></tr><tr><td rowspan="10">pH 6.8 buffer</td><td>YCW-MP</td><td>0.600</td><td>0</td><td>0.744</td><td>Diffusion</td></tr><tr><td>WP coated MP</td><td>0.799</td><td>0</td><td>0.949</td><td>Diffusion</td></tr><tr><td>ALG coated MP</td><td>0.776</td><td>0</td><td>0.847</td><td>Diffusion</td></tr><tr><td>WP coated YCW-MP</td><td>0.648</td><td>0</td><td>0.878</td><td>Diffusion</td></tr><tr><td>ALG coated YCW-MP</td><td>0.543</td><td>0</td><td>0.855</td><td>Diffusion</td></tr><tr><td>WP-YCW coated MP</td><td>0.557</td><td>0</td><td>0.717</td><td>Diffusion</td></tr><tr><td>ALG-YCW coated MP</td><td>0.548</td><td>0</td><td>0.715</td><td>Diffusion</td></tr></tbody></table>

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** *In vitro* trypsin and alpha-chymotrypsin inhibitory effect of particle (WP/ALG MP) and YCW-particle (YCW-MP) (n = 3). YCW-MP statistically different from MP: **p** < 0.01; ***p** < 0.001.</figcaption></figure>

insulin with the polymer matrix, rather than by a long-term shielding effect of YCW.

*Mucoadhesive properties of YCW-containing systems.* Mucoadhesion is expected to enhance local residence time at the absorptive surface and favor contact-driven uptake. The mucoadhesion of YCW-containing formulations was evaluated according to the method of (Rao and Buri, 1989), which is widely accepted for determining bioadhesive strength. The percentage of YCW-MP or YCW powder adhering to the intestinal mucosa was considered as the primary index of mucoadhesive capacity (Fig. 9). The results demonstrated that both MP and YCW-MP exhibited strong adhesion to the intestinal mucosa, with more than 90 % of particles remaining attached after 30 min of washing. YCW powder showed comparable adhesion to that of the bead formulations. Statistical analysis confirmed that no significant differences (p > 0.05) were observed among MP, YCW-MP, and YCW powder, whereas all formulations

<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Degradation studies of control: free-insulin (F-INS), INS loaded particle (INS-MP) and treatment: INS loaded in YCW-particle (INS-YCW-MP). Results are presented in percent of INS remaining undegraded, as mean ± standard deviation (n = 3). The experiments were performed in pH 7.4 buffer containing trypsin (10 IU/mL) and chymotrypsin (10 IU/mL) at 37 °C. INS remaining undegraded from INS-MP and INS-YCW-MP statistically different from F-INS: *p* < 0.05; **p** < 0.01; ***p** < 0.001 and INS remaining undegraded from INS-YCW-MP not statistically different from INS-MP: *p* > 0.05.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 9.** Percentage of control (glass beads and particle MP) and YCW-MP or YCW powder remaining attached on rabbit duodenum mucosa after washing with PBS for 30 min at an angle of 45°. Results are presented as mean ± standard deviation (n = 3 per group). Statistically different from glass beads: ***p** < 0.001. YCW powder and YCW/WP/ALG MP were not statistically different from MP: *p* > 0.05.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 10.** Cytotoxicity values (viability %) evaluated by trypan blue exclusion after incubation with free insulin (F-INS) at 300 µg/mL, free YCW solution (F-YCW), insulin loaded particle (INS-MP) and insulin loaded YCW particle (INS-YCW-MP) during 120 min (mean values ± SD; n = 3).</figcaption></figure>

significantly differed from the non-adhesive control (glass beads, ***p** < 0.001). These findings indicate that the intrinsic mucoadhesive nature of the alginate/whey protein matrix is already high, and the inclusion of YCW did not further increase adhesion under the test conditions.

The comparable adhesion of YCW powder and polymer-based beads
suggests that YCW contributes to the overall mucoadhesive behavior mainly through its surface biopolymers (β-glucans, mannoproteins, and chitin), which can form hydrogen bonds and electrostatic interactions with mucin glycoproteins. YCW contains small amounts of chitin, and chitosan—its deacetylated derivative—is well known for its mucoadhesive and biodegradable properties (Suknuntha et al., 2011). Several mechanisms, including electrostatic adsorption (hydrogen bonding, van der Waals interactions), wetting, diffusion, and fracture theories, have been proposed to explain the interaction between adhesive polymers and mucosal surfaces (Mortazavi and Smart, 1995; Dobrozsi et al., 1999). Although YCW did not significantly enhance adhesion beyond the alginate/whey protein baseline, its presence may help stabilize the bead–mucosa interface and could contribute biologically by modulating the local intestinal environment; YCW-derived polysaccharides (β-glucans, mannoproteins, chitin) are known to exhibit prebiotic and immunomodulatory activities that may favor local retention and uptake (Jofre et al., 2024).

### 3.4. Effect of YCW on insulin permeability in Caco-2 cells

**Cytotoxicity.** The cytotoxicity of YCW and YCW-MP is represented in Fig. 10. Viabilities were maintained in all cases close to 100 % after contact time of 2 h. YCW in DMEM solution were more toxic than YCW encapsulated in MP, certainly because YCW in solution had more contact with the monolayer than YCW in MP. Nevertheless, the overall

<figure><img src="image_13.png" /><figcaption>**Fig. 11.** (A) TEER % profiles as a function of time evaluated after control: no treatment (NT), unloaded particle (MP) and experiments: free-YCW (F-YCW) and YCW loaded particle (YCW-MP) (mean values ± SD; n = 3). TEER value of F-YCW, MP and YCW-MP statistically different from NT at 120 min: ***p < 0.001. TEER value of YCW-MP not statistically different from MP: p > 0.05. (B) Recovery % profiles of TEER values of Caco-2 cell monolayers after contact with MP, F-YCW, YCW-MP during 120 min and removal of the treatment. After 120 min application to Caco-2 monolayers, treatment was removed and epithelial cells were allowed to recover for 48 h in fresh cell medium, where the % of original TEER was monitored (mean values ± SD; n = 3).</figcaption></figure>

viability remained compatible with good biocompatibility for both the polymer matrix and YCW. These results confirm that the temporary modulation of epithelial permeability (see below) does not result from overt cytotoxic damage.

**Transepithelial electrical resistance (TEER).** Disruption of the epithelial cells was monitored by the TEER values evaluation which is representative of the tight junction complexes functionality between cells. When the tight junctions open, the TEER value will be reduced due to the water and ion passage through the paracellular route. At the beginning of the permeation experiments, the mean TEER value was 860 Ω.cm² (standard deviation ± 40 Ω.cm²) to indicate the junctional integrity of Caco-2 cell monolayers. Caco-2 cells monolayers were incubated for 120 min with no treatment (DMEM), with free YCW solution prepared in DMEM (F-YCW) or with MP containing (YCW-MP) or not YCW (MP) and suspended in DMEM (Fig. 11). In comparison with no treatment used as control, statistical difference decrease in TEER was observed after treatment addition (Fig. 11A). This result indicated that YCW and polymers had an impact on tight junction integrity. All the MP tested significantly decreased the TEER values. No significant difference was observed between MP and YCW-MP, suggesting that the WP/ALG matrix itself contributes to junction modulation, and that YCW does not dramatically amplify this effect under these conditions.

To evaluate the ability of cell monolayers to revert to their original impermeable state, TEER values were monitored over a 48 h period after removal apical samples and application of fresh cell culture medium. TEER values increased rapidly within 4 h to reach values comparable to baseline levels at 48 h for all the formulations (Fig. 11B). This reversible decrease in TEER, together with ~100 % cell viability, indicates that the permeability enhancement was transient and non-cytotoxic. These results suggest that YCW in solution or encapsulated in MP could potentially and temporary modify membrane permeability to promote momentarily drug absorption but didn't alter the monolayer integrity as suggested with trypan blue test. Several authors showed that a decrease in TEER values during exposure of Caco-2 cells to polymeric particles were related to an increase in INS permeability values (López and Peppas, 2004; Kavimandan et al., 2006).

**Insulin permeability.** The effect of YCW in solution (F-INS-YCW) or encapsulated in MP on the permeability of INS across Caco-2 monolayer is shown in Table 3. The transport of F-INS-YCW across Caco-2 cells was significantly increased (p < 0.001 at 120 min) compared to F-INS. The apparent permeability ($P_{app}$) values of F-INS-YCW ($P_{app}$ 11.75 ± 0.24 × 10⁻⁶ cm/s) were significantly superior to control (F-INS) ($P_{app}$ 7.27 ± 0.56 × 10⁻⁶ cm/s) (Table 3). In this way, TEER reductions, described above, were correlated with an increase in flux of INS across Caco-2 cell monolayers, indicating the capability of YCW to open the tight junctions. The important decrease of TEER profile observed with YCW was conceivably due to the capability of YCW to widen the paracellular route by interacting and interfering with junctional proteins of tight junctions as demonstrated for some polymers such as chitin (Sandri et al., 2010).

<caption>**Table 3**</caption>

Absorption of INS (in µg or %), mean apparent permeability ($P_{app}$) and absorption enhancement ratio (R) for INS across Caco-2 cell monolayers, in the presence of controls: free-INS (F-INS), INS loaded particle (INS-MP) and treatments for 120 min: free INS-YCW (F-INS-YCW), INS and YCW loaded particle (INS-YCW-MP) (mean values ± SD; n = 3).

<table><thead><tr><th rowspan="2">Sample</th><th colspan="2">Absorption</th><th rowspan="2">P<sub>app</sub> (x10<sup>-6</sup> cm.s<sup>-1</sup>)</th><th rowspan="2">R</th></tr><tr><th>µg</th><th>%</th></tr></thead><tbody><tr><td>F-INS</td><td>47.93 ± 3.66</td><td>16.45 ± 1.26</td><td>7.27 ± 0.56</td><td>1.00</td></tr><tr><td>F-INS-YCW</td><td>77.43 ± 1.56</td><td>26.56 ± 0.53</td><td>11.75 ± 0.24<sup>b</sup></td><td>1.62</td></tr><tr><td>INS-MP</td><td>63.00 ± 4.40</td><td>21.61 ± 1.51</td><td>9.56 ± 0.67<sup>a</sup></td><td>1.31</td></tr><tr><td>INS-YCW-MP</td><td>72.50 ± 2.24</td><td>24.87 ± 0.77</td><td>11.00 ± 0.34<sup>b,c</sup></td><td>1.51</td></tr></tbody></table>

<footnote><sup>a</sup> $P_{app}$ statistically different from F-INS, p < 0.05.</footnote>

<footnote><sup>b</sup> $P_{app}$ statistically different from F-INS, p < 0.001.</footnote>

<footnote><sup>c</sup> $P_{app}$ statistically different from INS-MP, p < 0.05.</footnote>
<caption>**Table 4**</caption>

Absorption of INS (in µg or %), mean apparent permeability ($P_{app}$) and absorption enhancement ratio (R) for INS across excised duodenum of rats, after 120 min, in the presence of controls: free INS at 300 µg/mL (F-INS), INS-particle (300 µg/mL, INS-MP) and treatment: INS-YCW-particle (300 µg/mL, INS-YCW-MP) (mean values ± SD; $n = 5$).

<table><thead><tr><th rowspan="2">Samples</th><th colspan="2">Absorption</th><th rowspan="2">P<sub>app</sub> (x10<sup>-6</sup> cm.s<sup>-1</sup>)</th><th rowspan="2">R</th></tr><tr><th>µg</th><th>%</th></tr></thead><tbody><tr><td>F-INS</td><td>10.75 ± 3.50</td><td>3.77 ± 1.23</td><td>3.30 ± 1.07</td><td>1.00</td></tr><tr><td>INS-MP</td><td>31.84 ± 2.15</td><td>11.15 ± 0.75</td><td>9.75 ± 0.66<sup>a</sup></td><td>2.96</td></tr><tr><td>INS-YCW-MP</td><td>35.10 ± 3.25</td><td>12.30 ± 1.14</td><td>10.74 ± 1.00<sup>a,b</sup></td><td>3.26</td></tr></tbody></table>

YCW but more certainly chitin would be able to disrupt epithelial tight junctions as described by Ranaldi et al. (2002).

Our results were in accordance with those of Fuller et al. (2007) who found that application of yeast suspensions resulted in a dose-dependent increase in Marker Protein HRP transported (Caco-2). ZO-1, actin and occludin proteins were demonstrated to be involved in yeast-induced tight junction opening (Fuller et al., 2007). The transport of INS was also greater when INS was encapsulated in YCW-MP with a significantly increased of the permeability. Our study had shown that YCW have high mucoadhesive strength. Due to the adherence of YCW to the mucosal tissues, INS transit time is delayed and the drug is localized to the absorptive surfaces. It improves drug bioavailability and effectiveness by facilitate paracellular transport via tight cell junction opening. In a study conducted by Gregorio et al., Limosilactobacillus fermentum ME-3, a probiotic with a yeast-like cell wall, was shown to protect Caco-2 monolayers from increased membrane permeability caused by irinotecan treatment (De Gregorio et al., 2023).

### 3.5. Ex vivo permeability in excised intestinal tissue

The *ex vivo* intestinal sac model (rat duodenum) includes a native mucus layer and endogenous proteases, which are not fully represented in the Caco-2 model. Absorption of free INS (F-INS) or INS encapsulated in MP containing (INS-YCW-MP) or not YCW (INS-MP) by rat duodenal sac was investigated *ex vivo* after 120 min treatment (Table 4). YCW-MP provided a protection against *ex vivo* enzymatic degradation but YCW didn't improve more INS absorption than WP/ALG MP. This differs from the Caco-2 results. The discrepancy between the *in vitro* Caco-2 results and the ex vivo data can be explained by fundamental differences between the models. In Caco-2 monolayers, the absence of mucus, the very small surface area, and the static environment create a confined system where YCW remain in direct and prolonged contact with the epithelium, resulting in measurable TEER reductions and increased insulin

<caption>**Table 5**</caption>

Pharmacological availability of INS in healthy rat, calculated after 120 min from plasmatic INS obtained after subcutaneous administration of free insulin (F-INS, 63 µg/rat) and duodenal administration of free insulin (F-INS, 730 µg/rat) or insulin loaded particle containing (INS-YCW-MP, 730 µg/rat) or not YCW (INS-MP, 730 µg/rat) (mean values ± SD; $n = 5$).

<table><thead><tr><th>Route</th><th>Sample</th><th>Dose (µg/rat)</th><th>AUC<sub>0-120 min</sub></th><th>Pharmacological availability PA (%)</th></tr></thead><tbody><tr><td>Subcutaneous</td><td>F-INS</td><td>63</td><td>7605 ± 95</td><td>100</td></tr><tr><td rowspan="2">Duodenal</td><td rowspan="2">F-INS</td><td rowspan="2">730</td><td>1943 ± 58<sup>b</sup></td><td>2.19</td></tr><tr><td>7845 ± 53<sup>a</sup></td><td>8.84<sup>a</sup></td></tr><tr><td>Duodenal</td><td>INS-MP</td><td>730</td><td>7853 ± 69<sup>a,c</sup></td><td>8.85<sup>a,c</sup></td></tr><tr><td>Duodenal</td><td>INS-YCW-MP</td><td>730</td><td></td><td></td></tr></tbody></table>

permeability. In contrast, native intestinal tissue contains a thick mucus layer, exhibits a complex 3-D architecture, and exposes the formulation to enzymatic activity. In the ex vivo duodenum sac, the formulation is distributed over a much larger surface area and diluted within the luminal fluid. Consequently, the local concentration of YCW reaching the epithelial tight junctions is greatly reduced, which limits their ability to modulate paracellular permeability. These differences could explain why the enhancement observed *in vitro* was not reproduced in native tissue or *in vivo*.

### 3.6. *In vivo* intestinal administration in healthy rats

To assess absorption *in vivo*, insulin formulations were administered directly into the rat duodenum rather than by oral gavage, because the ~1.4 mm beads cannot be swallowed safely and would not reliably pass the pylorus intact. The efficacy of INS-YCW-MP was assessed by a duodenal administration method, which has been used for determining absorption efficiency in animals (Table 5). Pharmacokinetic analysis over 120 min showed that the AUC<sub>0-120 min</sub> and the pharmacological availability of INS-YCW-MP duodenal administration was not significantly different from those of INS-MP duodenal administration ($p > 0.05$). Under these conditions, YCW did not significantly enhance systemic insulin exposure compared to beads without YCW. As in the *ex vivo* model, we attribute this to mucus layer, complex 3-D architecture, and enzymatic activity, which likely reduce the local concentration of YCW at the epithelial surface and limit its ability to modulate tight junctions *in vivo*.

### 3.7. Confocal evaluation of intestinal uptake

To visualize local delivery and confirm mucosal interaction, confocal microscopy was performed on duodenal sections collected 60 min after administration of INS-FITC-loaded YCW-MP (Fig. 12). The nuclei were stained using Hoechst stain and appeared blue, while the green fluorescence corresponds to INS-FITC. The substrates no treated (Fig. 12A) were characterized by no fluorescence while the tissue treated with INS-FITC-YCW-MP showed green spots into the epithelial mucosa close to the nuclei (Fig. 12B). Green fluorescent spots were also observed adherent to the mucosal surface, consistent with YCW-containing material in close contact with the epithelium. These images confirm (i) the mucoadhesive behavior of the beads and YCW, (ii) the proximity of released insulin to the absorptive epithelium, and (iii) the delivery of insulin in association with YCW at the intestinal surface.

Taken together, the *in vitro*, *ex vivo*, and *in vivo* data indicate that YCW (a) improves formulation properties (viscosity, insulin retention, enzyme inhibition), (b) enhances paracellular permeability *in vitro* by transient tight-junction modulation without cytotoxicity, and (c) promotes strong mucoadhesion. However, these advantages did not translate into a statistically significant improvement in systemic insulin exposure *in vivo*, likely due to dilution and dispersion effects in the intestinal lumen and the lack of an enteric protection strategy to deliver the beads intact beyond the stomach.

## 4. Conclusion

The present work investigated the incorporation of yeast cell wall into whey protein/alginate beads as a strategy to improve oral insulin delivery. YCW was successfully introduced either directly into the bead matrix or into the external coating. The presence of YCW increased the viscosity of the precursor formulations and limited insulin loss during the coating step, resulting in high encapsulation efficiencies (>98 %) and homogeneous insulin distribution within the beads.

All formulations exhibited rapid insulin release in simulated gastric and intestinal media. The addition of YCW did not significantly slow insulin release *in vitro*, and coated systems did not provide sustained release, indicating that the thin food-grade coatings used here act more

<footnote>a $P_{app}$ statistically different from control (F-INS), $p < 0.001$.</footnote>

<footnote>b $P_{app}$ not statistically different from INS-MP, $p > 0.05$.</footnote>

<footnote>a statistically different from control (F-INS, 730 µg/rat), $p < 0.001$.</footnote>

<footnote>b statistically different from control (F-INS, 63 µg/rat), $p < 0.001$.</footnote>

<footnote>c No statistically significant difference from INS-MP, $p > 0.05$.</footnote>
<figure><img src="image_14.png" /><figcaption>**Fig. 12.** Schematic representation displaying the composition of YCW-particle (YCW-MP) and their mechanisms for paracellular permeation. YCW-MP were instantaneously formed via the ionic gelation between ALG and WP molecules. The prepared mucoadhesive YCW-MP protected INS from the enzymatic degradation by inhibition of the intestinal enzymes. The YCW-MP also exhibited paracellular permeation enhancement throughout the entire small intestine due to the tight junction disruption. Confocal microscopic observations of the duodenum tissues treated during 60 min with (A) control (unloaded particle MP) and (B) INS-FITC-YCW-MP in duodenum of healthy male Wistar rats (scale bar represents 50 µm). Arrows referred to INS-FITC.</figcaption></figure>

as processing aids than as true diffusion barriers. Although Harland model fitting indicated that diffusion was the dominant release mechanism ($A > B$), our erosion measurements (matrix weight loss) showed that a certain degree of structural disintegration also occurred, particularly at pH 6.8, where beads exhibited significantly higher mass loss and diameter expansion. Therefore, the rapid release observed in vitro likely results from a combination of fast hydration, matrix relaxation and partial erosion, and not from pure diffusion alone. For this reason, we have refrained from concluding that no erosion occurred. YCW-containing beads inhibited the activity of intestinal proteases (trypsin and α-chymotrypsin) and, together with the WP/ALG matrix, were able to preserve a fraction of insulin from enzymatic degradation while maintaining immunoreactivity.

YCW and WP/ALG beads exhibited strong mucoadhesion to intestinal tissue, and YCW-containing formulations transiently and reversibly reduced transepithelial electrical resistance (TEER) in Caco-2 monolayers without causing cytotoxicity. This was associated with an increase in insulin permeability in vitro, supporting the role of YCW as a local permeability modulator at the epithelial surface.

However, these advantages did not fully translate to ex vivo intestinal sacs or to in vivo duodenal administration in rats, where insulin absorption from YCW-containing beads was not significantly higher than from beads without YCW. We attribute this discrepancy to the dilution and dispersion of YCW along the intestinal lumen, which likely reduces its local concentration and thus its ability to modulate tight junctions under physiological conditions.

Overall, our results indicate that YCW improves formulation properties (viscosity, retention of insulin during processing, enzyme inhibition, mucoadhesion, reversible tight junction modulation) and enhances insulin permeability in vitro, but does not yet lead to a measurable improvement in systemic insulin exposure in vivo. Future work should focus on (i) improving gastric protection and delivery to the intestine (e. g. enteric capsule or fluidized-bed gastroresistant coating), and (ii) using better-defined YCW fractions such as purified mannoprotein- and β-glucan-rich components, rather than heterogeneous commercial YCW powders, to achieve more consistent absorption enhancement.

## Formatting of funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

## CRediT authorship contribution statement

**Emmanuelle Lainé:** Writing – original draft, Methodology, Data curation.
**Valerie Hoffart:** Writing – review & editing, Writing – original draft, Supervision.
**Imen Dhifallah:** Writing – review & editing, Data curation.
**Ghislain Garrait:** Methodology, Data curation.
**Eric Beyssac:** Writing – review & editing, Supervision.

## Data availability

Data will be made available on request.

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