# Structural and mechanistic exploration in the development of floating drug delivery systems using calcium-pectinate gels with sodium bicarbonate as CO2 gas-forming agent

Worrawee Siripruekpong, Ruedeekorn Wiwattanapatapee, Odile Chambin, Ali Assifaoui

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Worrawee Siripruekpong, Ruedeekorn Wiwattanapatapee, Odile Chambin, Ali Assifaoui. Structural and mechanistic exploration in the development of floating drug delivery systems using calcium-pectinate gels with sodium bicarbonate as CO2 gas-forming agent. Journal of Drug Delivery Science and Technology, 2025, 107, pp.106729. 10.1016/j.jddst.2025.106729. hal-05143724

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# Structural and mechanistic exploration in the development of floating drug delivery systems using calcium-pectinate gels with sodium bicarbonate as CO₂ gas-forming agent

Worrawee Siripruekpong<sup>a,b</sup><sup>ID</sup>, Ruedeekorn Wiwattanapatapee<sup>a,b</sup>, Odile Chambin<sup>c,d</sup><sup>ID</sup>, Ali Assifaoui<sup>c,d,*</sup><sup>ID</sup>

## ARTICLE INFO

### Keywords:

- Gastroretentive systems

- Ionotropic gelation

- Pectin

- EGCG

- Anisotropic porous hydrogels

## ABSTRACT

This study used external ionotropic gelation to design floatable calcium-pectinate hydrogels incorporating (–)-Epigallocatechin-3-gallate (EGCG) as a model drug. Sodium bicarbonate at varying concentrations (0 %, 1 %, 3 %, and 5 % w/v) served as a gas-forming agent to produce carbon dioxide in simulated gastric fluid (SGF). The reaction between sodium bicarbonate and released protons during gelation formed a limited number of holes in the gel. Results showed the size and number of holes correlated with the 3D structure of pectin, with denser, rigid area forming smaller holes and softer areas forming larger holes. Moreover, the increased local pH from sodium bicarbonate led to EGCG degradation, especially in the softer parts of the gel. The dosage forms exhibited high encapsulation efficiency, with EGCG release in SGF following polymer matrix erosion and relaxation. Higher sodium bicarbonate concentrations accelerated EGCG release, highlighting the impact of CO₂ generation on drug release kinetics. This study demonstrates the potential of anisotropic gels as innovative drug delivery systems for controlled release in the stomach.

## 1. Introduction

Gastroretentive drug delivery systems are particularly suitable for drugs that exert their therapeutic effect in the gastric mucosa, such as in the management of *Helicobacter pylori* eradication and gastric ulcers [1]. These dosage forms are designed to remain to overcome physiological barriers by remaining in the stomach long enough to release the active pharmaceutical ingredient (API) in a controlled manner, extending drug release duration, reducing administration frequency, and improving patient compliance [2]. Various strategies have been employed to extend the gastric residence time, including the use of super-porous hydrogels, gas-generating systems, expandable systems, low-density

systems, and floating systems [3]. However, when the stomach empties its contents into the small intestine, the buoyancy of the dosage form can be hindered. To address this limitation, mucoadhesive polymers enable the dosage form to adhere to the mucosal lining of the stomach wall [2]. These polymers can be naturals, such as pectin, alginates, or semi-synthetics/synthetics, such as hydroxypropyl methyl cellulose (HPMC), Carbopol®934 [4].

Floating drug delivery system is a type of gastroretentive drug delivery system that has a lower density than the gastric fluid (~1.004 g/cm³) [5]. These systems can be classified into two types of buoyancy mechanisms: non-effervescent and effervescent systems [6]. In non-effervescent systems, a significant amount of swellable and

**Abbreviations:** (–)-Epigallocatechin-3-gallate, EGCG; Low methoxy pectin, LMP; Calcium carbonate, CaCO₃; Sodium bicarbonate or Sodium hydrogen carbonate, NaHCO₃; Calcium chloride dihydrate, CaCl₂·2H₂O; Sodium chloride, NaCl; simulated gastric fluid, SGF; Carbon dioxide, CO₂; Theasinensin A, TSA; (–)-Gallostechingallate, GCG; Thermogravimetric analysis, TGA; Scanning electron microscopy, SEM; one-way analysis of variance, ANOVA; three dimensional, 3D; Encapsulation Efficiency, EE.

* Corresponding author. L'Institut Agro, Université Bourgogne Europe, INRAE, UMR PAM, 21000, Dijon, France.

E-mail address: ali.assifaoui@ube.fr (A. Assifaoui).

<footnote><sup>a</sup> Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hatyai, Songkhla, 90112, Thailand</footnote>

<footnote><sup>b</sup> Phytomedicine and Pharmaceutical Biotechnology Excellence Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hatyai, Songkhla, 90112, Thailand</footnote>

<footnote><sup>c</sup> L'Institut Agro, Université Bourgogne Europe, INRAE, UMR PAM, 21000, Dijon, France</footnote>

<footnote><sup>d</sup> School of Pharmacy, Université Bourgogne Europe, 7 Bd Jeanne D'Arc, 21079, Dijon, France</footnote>
gel-forming polymers are used. When these polymers come into contact with gastric juices, they can either swell or form a gel, entrapping air within the gel structure. This action decreases their density, resulting in buoyancy or flotation [7]. In effervescent systems, gas-generating ex- cipients such as carbonate salts (CaCO₃ or NaHCO₃) and acidic in- gredients are used, which can generate carbon dioxide gas in the presence of gastric acid. This gas is then entrapped within a swellable hydrophilic biopolymer, enabling the dosage form to float and ensuring controlled release of the drug over an extended period. Recently, various types of floating drug delivery systems exist, including beads, micro- spheres, films, and tablets [8].

Pectin has gained considerable attention in the pharmaceutical for- mulations as thickening agent, gelling agent, or binding agent in tablet formulations [9]. This anionic polysaccharide, extracted from the cell walls of various plant sources such as citrus peels, apple pomace, and pomelo, consists of linear chains of α-(1,4)-linked d -galacturonic acid (GalA) and 1,2-linked L-rhamnose. Pectin are biocompatible and biodegradable polymers that form calcium-pectinate gels stable in acidic pH, which maintain the integrity of the dosage form in the stomach [10]. Their mucoadhesive properties enhance the gastric residence time, while the 3D network structure of the gel, with tunable mesh sizes, makes pectin a promising candidate for floating drug delivery systems [11]. Previous studies have involved acidifying the crosslinking solution by adding acetic acid to generate gas in the same time as the gelation process [12]. While this method enhanced buoyancy, it also risked weakening the 3D network formed by calcium-pectin, potentially lead- ing to significant drug loss. It was demonstrated that beads could not be formed when high sodium bicarbonate (10 % w/w) was used, because the gas release damaged the bead before the wall was sufficiently hardened [7]. In other studies, hydrogel beads were initially produced and subsequently washed before being either air-dried at 37 °C for 12 h or freeze-dried. In this process, the dissolved sodium bicarbonate crys- tallizes within the dried bead. The floating property appears when the dried beads come into contact with the gastric fluid. Furthermore, it was observed that air-dried beads exhibited a lower burst and slower release, whereas freeze-dried beads displayed considerably higher initial burst release with rapid release kinetics, likely due to their increased porosity

inducing by the drying process. Consequently, the highly porous beads demonstrated notable buoyancy ability and fast drug release [13].

Floating beads are produced through ionotropic “external” gelation (Fig. 1), where a solution containing low methoxy pectin (LMP), API, and sodium carbonate salt is dropped into a crosslinking solution of highly concentrated calcium chloride at room temperature. This causes immediate gelation on the surface of the droplets, forming insoluble beads (Fig. 1a). Such external gelation process displays anisotropic properties, with pectin concentration and the Young’s modulus decreasing from the bead’s outer periphery to its core (Fig. 1). This gradient corresponds to an increase of the mesh-size of the 3D network [14]. However, the distribution to the API and the gas-generating salt within the bead is still under investigation. To examine this distribution, a cylindrical calcium-pectinate hydrogel was produced by the external unidirectional diffusion of calcium ions into the pectin solution (Fig. 1b). Both the pectin and calcium solutions were separated by a dialysis membrane. The gel part close to the dialysis membrane, named “NEAR,” behaves as the periphery of the bead, while the gel part farther from the membrane, named “FAR,” behaves as the core of bead [15].

This work focuses on calcium-pectinate cylindrical gels obtained by external gelation, incorporating sodium hydrogen carbonate (NaHCO₃) as the gas-generating and (−)-Epigallocatechin-3-gallate (EGCG) as the drug model. (−)-Epigallocatechin-3-gallate (EGCG), the primary active compound in green tea, holds various health benefits including antiox- idant [16], antiulcer [17], anti-obesity [18], anti-aging [19], and po- tential anti-cancer [20] properties. Despite its advantages, EGCG faces challenges in oral administration due to its hydrophilic nature, low bioavailability [21], short half-life, low permeability and poor stability [22]. EGCG serves as a model drug in a floating gel formulation, aiming to overcome these problems and to enhance drug bioavailability. The main aim of this work is to study the behavior, both in terms of location and concentration, of the EGCG and NaHCO₃ components within the calcium-pectinate gel. This investigation was carried out to understand the formation of holes, essential elements for the buoyancy of the beads. Previous studies have shown that the mesh-size in the near part of polygalacturonate gel was about 7.5 nm closely aligned with the poly- mer's persistence length (Lp) [23]. Therefore, the mesh-size appears to

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** External gelation of calcium-pectinate gels. For hydrogel beads (a) the diffusion of calcium ions is radial, while in cylindrical gels (b), it follows the z-axis. The regions closer to the dialysis membrane “NEAR” (a) or the periphery of the bead (b) exhibit higher pectin concentration and Young’s modulus. Conversely, the opposite trend is observed in the region farther away from the dialysis membrane, referred as the "FAR" region or the core of the bead.</figcaption></figure>
gradually increase from the near to the farthest part of the gel [23]. Calcium-pectinate hydrogels with and without EGCG were designed and various amounts of sodium bicarbonate (0, 1 %, 3 % and 5 % w/v) were examined. The obtained gels were sliced into three parts noted “NEAR”, “MIDDLE” and “FAR” (Fig. 1b). For each gel slice, the amount of organic (pectin and EGCG) and inorganic (sodium, calcium ions ...) compounds were determined using thermal gravimetry analysis (TGA), the Young’s modulus was measured by stress-strain tests, the microstructure was observed by scanning electronic microscopy (SEM) and EGCG release was assessed by using in-vitro dissolution tests.

## 2. Materials

(-)-epigallocatechin gallate (EGCG) was purchased from Xi'an Nate Biological Technology Co., Ltd (Xian-Beilin, China). Low methoxy pectin (LMP; UNIPECTINE™, OF 300C) was a gift from Cargill (Baupte, France). The degree of esterification (DE) for low-methyl pectin (LMP) ranges from 27 % to 33 % and a mean molecular mass of 174 ± 9 kDa [24]. Sodium hydrogen carbonate (NaHCO₃) was obtained from Cooperatoration Pharmaceutique Française, Co, Ltd, Melun, France. Calcium chloride dihydrate (CaCl₂, 2H₂O) was used as an ionic cross-linker. Sodium chloride (NaCl) was applied to maintain and balance isotonic conditions. Calcium chloride and sodium chloride were purchased from Sigma - Aldrich Co, Ltd (Saint-Louis, Missouri, USA) and were of analytical grade. Distilled water was used in this study. All other reagents used were of analytical grade or pharmaceutical grade.

## 3. Methods

### 3.1. Preparation of pectin solutions

Three grams of LMP powder was dissolved in approximately 100 mL of sodium chloride solution (10 mM) for 2–4 h. Once achieving complete dispersion and obtaining a homogeneous colloidal solution, the pH was then adjusted to 5.5, to achieve a totally negatively charged polysaccharide, by adding 1 M sodium hydroxide solution (NaOH). LMP solution was dialyzed against sodium chloride solutions (100 mM) for 2 h (2 times) and 16 h, by using a dialysis membrane tubing (Snakeskin™, Thermo-Fisher Scientific, Regenerated Cellulose, Molecule cut-off 3.5 kDa). This was achieved to eliminate free ions and unbound small molecules (sugar and proteins) from the LMP solution. The volume ratio of the sodium chloride and polymer solution was 10:1. Finally, the volume of the mixture was adjusted to 100 mL in order to obtain a final concentration of LMP of 3 % w/v [23].

### 3.2. Preparation of macroscopic cylindrical floating gels

Anisotropic floating gels were performed using the external gelation method which accord with the diffusion of calcium ions from an external solution into the pectin solution. In this work, we studied mainly the macroscopic cylindrical gels (Fig. 1b). These gels are used to encapsulate (-)-epigallocatechin gallate (EGCG). Therefore, various amounts of sodium bicarbonate powder (NaHCO₃) were dissolved in a 3 % w/v low-methoxy pectin (LMP) solution, where 1 % w/v of EGCG had been previously solubilized. Four concentrations of NaHCO₃ were tested (0 %, 1 %, 3 %, and 5 % w/v). Macroscopic cylindrical gel without EGCG and sodium bicarbonate was also prepared as a control sample. Therefore, 5 mL of the mixture, which included LMP alone, LMP + NaHCO₃ or LMP + NaHCO₃+EGCG, was poured into a cylindrical glass tube (21 mm diameter and 150 mm height). It must be noted that the amount of EGCG in each loaded hydrogel is equal to 50 mg. The lower end of the tube was covered by a hydrated dialysis membrane (SnakeSkin®; Molecular weight cut-off = 3.5 kDa) to create a barrier between the polymer solution and the cation reservoir. Subsequently, the lower part of this tube was immersed in 20 mL of calcium chloride solution (100 mM) at room temperature for 12 h. The resulting cylindrical gel was then cut into

three or four horizontal slices. The slice closest to the dialysis membrane was referred as “NEAR”, while the farthest slice was denoted as “FAR”. Cylindrical gels with and without EGCG were designated as CGE and CG, respectively. In instances where 1 % of NaHCO₃ was utilized, the gel containing EGCG was labeled CGE_1 %. The composition of the different formulations can be found in table S1. After obtaining the hydrogels, calcium chloride solutions were collected and both the pH and the non-encapsulated EGCG were measured, respectively. Finally, the slices of hydrogel were dried at 40 °C and 30 % relative humidity for 24 h. The dried forms were then stored in a tight container at room temperature before use.

### 3.3. Physicochemical properties of cylindrical gels

#### 3.3.1. Macroscopic observations, weight and density determination

The morphology and the shape of both fresh and dried cylindrical dosage forms were assessed using the optical microscope equipped with a ruler. The diameter and the thickness of each gel slice (NEAR, MIDDLE, and FAR) were measured at least on three dosage form formulations. The results for diameter and thickness were presented as an average and standard deviation (SD). The mass of each slice was recorded before and after the drying process. The density was calculated from the total mass of the dosage form divided by its volume, which was calculated based on the diameter and thickness of the cylinder. The claimed density of floating gels was less than the density of the simulated gastric fluid (1.004 g/mL) [25].

#### 3.3.2. Floating behaviors

Cylindrical gels were separated into 3 parts such as NEAR, MIDDLE, and FAR. Each fresh gel slice was observed concerning floating behavior in 20 mL of 0.1 N hydrochloric acid solution (pH = 1.2). The total floating time was the duration of time that gels can maintain at the surface of the acidic media [26]. The optimized total floating time should be more than 4 h to show the floatability of gels. The buoyancy property of floating gels was recorded at t = 0, 24, and 48 h.

#### 3.3.3. Mechanical properties of cylindrical gels

The tensile strength test was a method to analyze mechanical properties by using a texture analyzer (Brookfield) in the Texture Pro CT program. Young's modulus was mechanical properties described as the elongation or compression of the object when exposed to a force. This test was provided with plate geometry with a 1.5 kg load cell. Each fresh cylindrical gel slice was compressed at a speed of 0.1 mm/s until 30 % strain of the sample [15]. It should be set to zero before testing. All samples were immediately tested after the gel formation in order to avoid dehydration. All formulations were tested in triplicate. The curve stress as a function of the strain allows the determination of the Young's modulus.

#### 3.3.4. Encapsulation efficiency

Each fresh gel slice was introduced in 20 mL of 0.1 N hydrochloric acid solution (pH = 1.2) and was grinded and homogenized using Ultra-Turrax® Tube Drive; ST Tube (IKA® Werke GmbH&Co.KG, Staufen, Germany) at the maximum speed (6000 rpm) for 10 min. The obtained mixture was filtered through Whatman® (No. 1, diameter 110 mm). EGCG concentration was determined by UV Spectrophotometer at the wavelength (λmax) = 274 nm [27]. The encapsulation efficiency was calculated with the following equation:

$$ \%EE = (E / E_t) \times 100 $$

Where E is the actual total amount of EGCG encapsulated into the dosage form.

$E_t$ is the theoretical amount of EGCG added.
### 3.4. In-vitro dissolution studies

The dissolution studies were performed using a rotating paddle apparatus type II within a dissolution tester, operating at 50 rpm and with a temperature of 37 ± 0.5 °C. Each gel slice (fresh or dried) (NEAR, MIDDLE, and FAR) was immersed in 200 mL of simulated gastric fluid (0.1 N hydrochloric acid; pH = 1.2). At specific intervals (t = 30, 60, 120, 180, 240, 300, 360, 420, and 480 min), samples (5 mL) were collected and replaced with an equivalent volume of fresh acidic media. The quantity of EGCG was assessed using UV Spectrophotometer at the wavelength 274 nm. EGCG concentration was determined from the absorbance by using a standard curve (Figure S1). The dissolution test experiment was repeated in triplicate. The cumulative drug release percentage was plotted as a function of time. The reported results represent the average values ± standard deviation. The release kinetics was fitted by using Kormeyer - Peppas model [28,29].

$$ \text{Kormeyer - Peppas models} = kt^n $$

Where M<sub>t</sub> is the amount of EGCG released at time t, M is the total amount of EGCG released at infinite time (complete release), n is the kinetic constant, and t is the release exponent, which indicates the mechanism of drug release.

n = 0.5 indicates Fickian diffusion-controlled release

< n ≤ 1.0 indicates non-Fickian transport or anomalous transport

n = 1.0 indicates zero-order release

n > 1 indicates super case II transport

### 3.5. Thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) was used to quantify the sodium bicarbonate within each gel slice (NEAR, MIDDLE, and FAR). Given the high moisture content of these gels (95–98 %), initial drying was

conducted to reduce the water content significantly. This step aimed to enhance the distinction in material loss during heating. TGA analysis (Mettler Toledo TGA, Swiss) was conducted under a controlled airflow, subjecting the samples to a heating rate of 20 °C/min, gradually raising the temperature from room temperature to 800 °C. Prior to analysis, samples were ground and approximately 20 mg of each was weighed into the sampling pan. Each sample underwent triplicate testing for accuracy [30].

### 3.6. Scanning electron microscopy (SEM)

Morphological study of the floating gels was examined by scanning electron microscopy (SU 3800, HITACHI, Hitachi High-Tech Corporation, Co, Ltd, Japan). Cylindrical gels were dried by hot air oven method at temperature 40 °C for 12 h before analysis. Scanning electron microscopy (SEM) imaging was used to observe morphological characteristics of dosage forms. These obtained cylindrical gels were coated with a gold thin layer at the operating voltage of 20 keV under a vacuum.

### 3.7. Statistical analysis

The results were reported as average values ± standard deviation. The data were analyzed using a one-way analysis of variance (ANOVA) processed by using the SPSS program to determine if the differences among mean values were significant. Statistical significance was defined at p-values less than 0.05.

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Physical appearance of calcium pectinate gels without EGCG (a) and with EGCG (b). (c) illustrates the three slices (NEAR, MIDDLE, and FAR) of the gel CGE_5 %.</figcaption></figure>
# 4. Results

## 4.1. Macroscopic observation of calcium pectinate gels

Fig. 2 illustrates cylindrical gels obtained at four different concentrations of sodium bicarbonate, both with and without EGCG. In all formulations, the near part of the gel seems to be harder than the far part of the gel. This discrepancy arises from the “external” gelation process, allowing the creation of a large gradient of cross-linked polymer concentration along the vertical direction (z-axis). Consequently, the near part from the cation reservoir expelled water, leading to a higher polymer concentration compared to the farthest part [14,31].

In the absence of EGCG, the gels exhibited slight turbidity, which intensified with an increase in the amount of NaHCO₃. The observed turbidity in these hydrogels was likely due to the emergence of mesoscopic heterogeneities scattering light (typically 1–10 µm size) [23]. The inclusion of EGCG (CGE_0 %) in the gel maintained the slight turbidity with a subtle color shift toward a pinkish hue. However, the presence of sodium bicarbonate significantly altered the gel’s color, displaying a gradient from grey in the near parts of the gels to dark brown in the farthest part of gel (Fig. 2c). The change in color was due to the degradation of EGCG. Surprisingly, this degradation depended on the positioning of EGCG within the gel. In the near part, EGCG seemed to keep its native structure more than in the farthest parts. This degradation may be due to the variation of the pH inside the gel. It is hypothesized that the pH was higher in the farthest parts compared to the nearest part, possibly due to an assumed gradient of sodium bicarbonate within the gel. The gelation of pectin in the near part resulted in the expulsion of water and sodium bicarbonate [14].

The mass, thickness, and diameter of both cylindrical gels, with and without EGCG, were measured (refer to Table S1). It was observed that the total mass of the gel increased with higher amount of sodium bicarbonate. Specifically, the mass increased from 4.40 to 5.15 g for CGE_0 % and CGE_5 %, respectively and from 4.56 to 5.18 g for CGE_0 % and CGE_5 %, respectively. Additionally, the thickness of the gel increased with the addition of sodium bicarbonate. Using these measurements, the volume and density of the gel were determined (refer to Table S1). The density was found to decrease from 0.95 to 0.90 g/cm³ for CGE_0 % and CGE_5 %, respectively; and from 0.99 to 0.87 g/cm³ for CGE_0 % and CGE_5 %, respectively. The density of the gel decreased with the increase of sodium bicarbonate. This suggests that CO₂ gas forms within the gel during the gel formation as depicted in Fig. 2c. A part of sodium bicarbonate may interact with protons released when carboxyl (COOH) groups of GalA interacting with the crosslinker agent (Ca²⁺). Moreover, the gel’s density was lower than that of gastric fluid (~1.00–1.01 g/cm³). When these hydrogels were introduced into gastric fluid with a pH of 1.2, the interaction between sodium bicarbonate and H⁺ lead to increased CO₂ gas production. This interaction resulted in a more porous gel with lower density, thereby enhancing its buoyancy [22]. The gels, both with and without EGCG, were cut into three equivalent slices (NEAR, MIDDLE, and FAR) and weighed. The resulting masses and percentages in each slice are presented in Table S2. Across all samples, the mass consistently decreased from the near to the far region, whatever the initial composition of the gel. This decline was attributed to polymer accumulation in the near part due to the external gelation process [14].

## 4.2. Mechanical properties of the hydrogels

Fig. 3 illustrates the variation of Young’s modulus (E) of the three gel slices without EGCG (NEAR, MIDDLE, and FAR) as a function of the initial amount of sodium bicarbonate (NaHCO₃). The results indicated that Young’s modulus was consistently higher in the near part of the gel compared to the farthest part, regardless of the sodium bicarbonate concentration (insert in Fig. 3). The elevated Young’s modulus in the near part was attributed to the polymer accumulation in the near part

<figure><img src="image_6.png" /><figcaption>Fig. 3. Young’s modulus of each gel slice without EGCG (NEAR, MIDDLE, and FAR) as a function of the concentration of sodium bicarbonate added. The insert figure represents the evolution of Young’s modulus for CG_0 %, CG_1 %, CG_3 %, and CG_5 % for the three gel slices.</figcaption></figure>

during the gelation process [32]. It is admitted that Young’s modulus (E) is directly correlated with the polymer chain concentration [LMP] according to equation E = a [LMP]² [33].

Furthermore, the introduction of sodium bicarbonate led to a reduction in Young’s modulus for all three slices (Fig. 3). This decrease could be linked to the generation of CO₂ gas during the gelation process, potentially explaining the observed lower density in gels with high sodium bicarbonate amounts (Table S2). Notably, Young’s modulus (E) is inversely proportional to the mesh size (ξ) of the gel according to the molecular theory of rubber elasticity [34] according to the following equation:

Where T, R, and N<sub>A</sub> are the temperature, the gas constant, and the Avogadro number, respectively. The density of crosslinks (ρ<sub>CL</sub>) is given by E/3RT.

Consequently, the observed decrease in Young’s modulus with an increase in sodium bicarbonate may be attributed to an increase in the mesh-size in the gel network. It must be noted that the presence of EGCG in the gel didn’t affect significantly the behavior of Young’s modulus in relation with sodium bicarbonate concentration (Figure S8).

## 4.3. Encapsulation efficiency of EGCG in cylindrical gel formulations

The encapsulation efficiency (EE) of EGCG in the gels, at different concentrations of sodium bicarbonate (0, 1, 3, and 5 %) and for the corresponding gel slices (NEAR, MIDDLE, and FAR) is presented in Table 1. During the gelation process, the EGCG loss in the crosslinking solution was around 9 % for CGE_0 % and it diminished with the increase in the initial sodium bicarbonate amount. Therefore, the encapsulation efficiency (EE) was notably high (>84.7 %). This elevated value was due to the gel densification in the near part, leading to a reduction in mesh size. Notably, the formulations CGE_3 % and CGE_5 % exhibited the highest EE (93.5 %). Furthermore, there was a decline in EE from the near to the farthest part of the gel, indicating the presence of a gradient of EGCG within the gel.

## 4.4. Floating behaviors of the cylindrical gel

The floating ability of calcium pectinate gels as a function of the amount of sodium bicarbonate in SGF was determined in Table 2. Floatability depended on the density of the gel which must be lower than the density of the SGF (~1.004 g/cm³) [5]. According to Table S1, it was found that the density of all the gels was ranged from 0.87 to 0.95. Such
<table><caption>**Table 1**
the percentage of EGCG loss during the gelation process and the encapsulation efficiency of EGCG in different slices of the gel (NEAR, MIDDLE, and FAR).</caption><thead><tr><th rowspan="2">Formulation</th><th colspan="3">EGCG released in the crosslinking solution</th><th colspan="3">% Encapsulation Efficiency (EE) in each slice</th><th rowspan="2">% EE in the whole gel</th></tr><tr><th>Quantity (mg)</th><th>%</th><th></th><th>NEAR</th><th>MIDDLE</th><th>FAR</th></tr></thead><tbody><tr><td>CGE_0 %</td><td>4.5</td><td>9.0 %</td><td></td><td>86.7 ± 0.1 %</td><td>84.7 ± 0.6 %</td><td>82.7 ± 0.1 %</td><td>84.7 ± 1.3 %</td></tr><tr><td>CGE_1 %</td><td>3.1</td><td>6.3 %</td><td></td><td>91.0 ± 0.1 %</td><td>88.5 ± 0.1 %</td><td>83.6 ± 0.1 %</td><td>87.7 ± 3.1 %</td></tr><tr><td>CGE_3 %</td><td>1.8</td><td>3.6 %</td><td></td><td>94.9 ± 0.2 %</td><td>93.7 ± 0.2 %</td><td>92.2 ± 0.9 %</td><td>93.6 ± 1.1 %</td></tr><tr><td>CGE_5 %</td><td>1.4</td><td>2.9 %</td><td></td><td>96.7 ± 0.3 %</td><td>93.9 ± 0.4 %</td><td>90.0 ± 0.5 %</td><td>93.5 ± 2.7 %</td></tr></tbody></table>

<table><caption>**Table 2**
Floating behaviors of cylindrical gel slices (NEAR (N), MIDDLE (M), and FAR (F)) when the formulation is introduced in a simulated gastric fluid (pH = 1.2) and room temperature. Cross (X) indicates that the gel slice didn't float.</caption><thead><tr><th rowspan="3">Formulations</th><th colspan="12">Floating behavior of dried gels</th></tr><tr><th colspan="3">Start time</th><th colspan="3">t = 24 h</th><th colspan="3">t = 48 h</th><th colspan="3">t = 72 h</th></tr><tr><th>N</th><th>M</th><th>F</th><th>N</th><th>M</th><th>F</th><th>N</th><th>M</th><th>F</th><th>N</th><th>M</th><th>F</th></tr></thead><tbody><tr><td>CG_0 %</td><td>✓</td><td>✓</td><td>✓</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td></tr><tr><td>CG_1 %</td><td>✓</td><td>X</td><td>✓</td><td>X</td><td>X</td><td>✓</td><td>X</td><td>X</td><td>✓</td><td>X</td><td>X</td><td>X</td></tr><tr><td>CG_3 %</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td></tr><tr><td>CG_5 %</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>X</td><td>X</td><td>✓</td><td>X</td><td>X</td><td>X</td></tr><tr><td>CGE_0 %</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td></tr><tr><td>CGE_1 %</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>X</td><td>X</td></tr><tr><td>CGE_3 %</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>CGE_5 %</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr></tbody></table>

a low density allowed the different gel slices to float when they were added to SGF. It was observed that both the whole gels and the corresponding slices floated in SGF during 48 h when more than 3 % of sodium bicarbonate was used (Fig. 4).

### 4.5. *In vitro* dissolution test of floating gels

The results of *in-vitro* dissolution studies of EGCG from calcium pectinate gels at various concentrations of sodium bicarbonate (0 %, 1 %, 3 %, 5%w/v) are presented in Fig. 5. Across all formulations, the release rate of EGCG was higher in the farthest part of the gel compared to the near part. This difference can be attributed to the larger mesh-size in the farthest part compared to the nearest part. After 8 h of dissolution, EGCG release in the three gel slices ranged from approximately 40%–60 % for CGE_0 % and CGE_1 %. In the case of CGE_3 % and CGE_5 %, EGCG released after 8 h was around 60 % in the near part; while in the far part, EGCG release was equal to 70 % and 93 % for CGE_3 % and CGE_5 %, respectively. The dissolution curves were fitted by using Korsmeyer–Peppas. It was found that n values in Korsmeyer–Peppas's model varied from 0.18 to 0.40 indicating that the release rate is predominantly controlled by erosion and relaxation of the polymer matrix, rather than by simple Fickian diffusion.

### 4.6. Characterization of dried hydrogels

The obtained hydrogels were then dried at 40 °C and 30 % relative humidity for 24 h. The microstructure of the dried hydrogels was then observed by scanning electron microscopy (SEM). The repartition of

<table><tr><td>CG_5%</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>X</td><td>X</td><td>✓</td></tr><tr><td>CGE_0%</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>X</td><td>X</td><td>X</td></tr><tr><td>CGE_1%</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>X</td></tr><tr><td>CGE_3%</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr><tr><td>CGE_5%</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td><td>✓</td></tr></table>

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Photos of gel slices (NEAR, MIDDLE, and FAR) of CGE_5 % in SGF (pH = 1.2; room temperature) after 48 h.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Fig. 5. *In-vitro* dissolution test of EGCG in simulated gastric fluid (pH = 1.2, T = 37 °C) for CGE_0 % (a), CGE_1 % (b), CGE_3 % (c) and CGE_5 % (d). For each formulation, the three gel slices: NEAR (N), MIDDLE (M), and FAR (F) were tested.</figcaption></figure>

organic and inorganic components (or parts) was also determined by thermal gravimetric analysis (TGA).

### 4.6.1. Thermogravimetric analysis (TGA) of dried gels

Thermogravimetric analysis (TGA) of dried gels (CGE_0 %, CGE_1 %, and CGE_5 %) along the corresponding slices (NEAR, MIDDLE, and FAR) is presented in Figure S3. Table 2 provides the percentage of organic and inorganic materials in the three slices. Three distinct thermal phases were observed for each dried slice. The first thermal event occurred in the temperature ranges of 100–150 °C, attributed to the weight loss from evaporating water. The second thermal event, ranging from 150 °C to 500 °C, was related to the decomposition of organic compounds mainly low methoxyl pectin and EGCG. Finally, a plateau was observed up to 500 °C, associated to the non-degraded inorganic material (sodium chloride, calcium chloride, and sodium carbonate). It is important to note that the dissolved sodium bicarbonate inside the gel crystallized during the gelation process [35]. Therefore, thermal decomposition of sodium bicarbonate (NaHCO₃) released H₂O(g), CO₂(g), and Na₂CO₃(s) [36]. Water vapor and carbon dioxide contribute to the first and second thermal events, respectively. Meanwhile, the remaining sodium carbonate (Na₂CO₃) contributed to the final plateau observed up to 500 °C. It must be noted that the thermal decomposition of Na₂CO₃ into Na₂O and CO₂ occurred at a temperature higher than 850 °C [37]. Unfortunately, this decomposition could not be observed as the analysis was stopped at 800 °C. As expected, the plateau observed in the third event (sodium carbonate) was more pronounced when the initial quantity of NaHCO₃ was introduced (Table 2). Moreover, it was observed an increase in the percentage of the remaining matter up to 500 °C from the near to far part (Fig. 6). TGA results indicated clearly that the concentration of sodium bicarbonate within the gel was high in the farthest part of the gel. This high concentration can explain the alteration in the gel color (Fig. 2), attributed to the EGCG degradation due to an increase of the pH [38] in the farther slices of the gel.

### 4.6.2. Scanning electron microscopy (SEM)

SEM observations were conducted on dried hydrogels, analyzing the three slices (near, middle, and far) for each formulation (Fig. 7). After

<figure><img src="image_9.png" /><figcaption>Fig. 6. the percentage of material remaining after the thermal treatment up to 500 °C.</figcaption></figure>

drying process, the gel which contains 95 % of water content, loses its three-dimensional structure created by the pectin network. The resulting structure of the dried hydrogel appeared as relatively transparent, hard films (see Figure S9). Notably, white dots on the surface of these films, indicated by thick arrows in Fig. 7, were likely attributed to NaHCO₃ crystals formed during the drying process [35]. For CGE_5 % dried gel, clear evidence of hole formation was observed, indicating the potential generation of CO₂ during the gelation process. This phenomenon may be attributed to the interaction between the carboxylic groups and the calcium, leading to the release of protons that interacted with NaHCO₃ in wet state. Furthermore, when introduced into simulated gastric fluid (pH = 1.2), the CGE_5 % dried gel displayed varying size and quantities of holes as detailed in Table 3.
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** SEM images for each dried slice (NEAR, MIDDLE, and FAR) gels (CGE_0 %, CGE_1 %, CGE_3 % and CGE_5 %). The thick arrows point out NaHCO₃ crystals formed during drying the gel. The dotted arrows indicated the formation of holes. Images in the right side depicted the CGE_5 % gel that was introduced in SGF to induce CO₂ generation.</figcaption></figure>

<table><caption>**Table 3**
percentage of water, organic and inorganic material present in the different slices of the dried hydrogels obtained from TGA data (CGE_0 %, CGE_1 %, and CGE_5 %).</caption><thead><tr><th rowspan="2">Formulation</th><th rowspan="2">PART</th><th>First and second thermal events</th><th>Third event</th></tr><tr><th>Water and organic material</th><th>Sodium and calcium</th></tr></thead><tbody><tr><td rowspan="3">CGE_0 %</td><td>NEAR</td><td>91 %</td><td>9 %</td></tr><tr><td>MIDDLE</td><td>81 %</td><td>19 %</td></tr><tr><td>FAR</td><td>82 %</td><td>18 %</td></tr><tr><td rowspan="3">CGE_1 %</td><td>NEAR</td><td>76 %</td><td>24 %</td></tr><tr><td>MIDDLE</td><td>77 %</td><td>23 %</td></tr><tr><td>FAR</td><td>70 %</td><td>30 %</td></tr><tr><td rowspan="3">CGE_5 %</td><td>NEAR</td><td>71 %</td><td>29 %</td></tr><tr><td>MIDDLE</td><td>61 %</td><td>39 %</td></tr><tr><td>FAR</td><td>53 %</td><td>47 %</td></tr></tbody></table>

## 5. Discussion

The external ionotropic gelation involves the diffusion of cross-linking cations (Ca²⁺) from an external reservoir into the pectin solution. These gels can be obtained by placing the pectin solution in contact with a high concentrated calcium chloride solution, resulting in bead formation (Fig. 1a), or by allowing the crosslinking agent to diffuse through a dialysis membrane, leading to the formation of cylindrical gels (Fig. 1b). In both cases, the resulting gel exhibits an anisotropic behavior, where the region close to the dialysis membrane (or the periphery of the bead) is denser and has a low mesh size. This is attributed to the accumulation of pectin chains during the gelation process [14]. In contrast, the region of the gel farthest from the dialysis membrane (or the core of the bead) was less dense and exhibited a large mesh size.

In this study, these anisotropic gels were utilized as a dosage form targeting API (EGCG) release in the stomach, aiming to prolong its residence time. Therefore, sodium bicarbonate, acting as CO₂ gas generator, was introduced in the pectin solution before gelation. The resultant gel, when in contact with gastric fluid, will generate CO₂, thereby reducing the density of the dosage form and providing buoyancy.

Moreover, the mucoadhesivity of the pectin may further extend residence time by promoting the adhesion of the dosage form to the mucosal stomach wall [2].

Calcium-pectinate gels (CGE_0 %) had an initial density of approximately 0.99. This density decreased with the increase of the amount of sodium bicarbonate and reached a value of 0.87 for CGE_5 %. The reduction in density can be attributed to the formation of holes as illustrated in Fig. 2c. These holes, resulting from the generation of CO₂, emerged when dispersed sodium bicarbonate within the gel reacted with protons in the presence of water. Protons are potentially released as two carboxylic groups of the pectin interact with calcium ions following to the bidentate model [39]. It is important to highlight that only a small amount of sodium bicarbonate may react with the released protons, given the excess of sodium bicarbonate introduced initially. Therefore, the distribution of sodium carbonate within the gel is non-uniform, with its concentration increasing from the near to the farther part of the gel. This hypothesis gains support from the observed alteration of the EGCG color (Fig. 2). Specifically, in the near part of gel, the pH appeared to be low, maintaining the natural color of EGCG (light grey). In the contrast, in the farther part of the gel, the color of the gel became darker, confirming the degradation of EGCG due to the increased pH. Epigallocatechin gallate (EGCG) can be unstable and undergo degradation in solution through processes such as epimerization and auto-oxidation [40]. The degradation of EGCG results in a color change from beige to brown, indicating the polymerization of EGCG dimers and transformation into theasinensin A (TSA) under basic condition (pH > 5.5). TSA is a polyphenolic compound known for its anti-inflammatory activity by suppression pro-inflammatory mediators [41]. The epimerization of EGCG to (–)-Gallocatechingallate (GCG) occurs in the pH range of 2–5.5. GCG is a trans-epimer catechin with non-toxic and biological activities similar to its cis form. Catechin epimerization is a reversible process and does not alter the anti-inflammatory and antioxidant activities of both EGCG and GCG. Therefore, the color change of EGCG indicated the pH changes due to the presence of sodium bicarbonate inside hydrogels. The non-uniform distribution within the gel can be confirmed by SEM observation (Fig. 7) and thermogravimetric analysis (Table 2). SEM reveals the presence of NaHCO₃ crystals in the
dried hydrogel [35] and TGA indicated that the quantity of this material increased from the near to the farther part of the dried gel (Fig. 6). Moreover, the formation of holes inside the gel of calcium-pectinate contributed to the decrease of the Young's modulus and therefore an increase of the mesh size of the 3D -network of calcium-pectinate (Fig. 3).

In summary, a limited amount of NaHCO₃ contributed to the formation of holes within the gel, while a significant quantity of sodium bicarbonate remained distributed inside the gel with a gradient in concentration. In contact with the simulated gastric fluid (pH = 1.2), the remaining NaHCO₃ reacted with protons of SGF, creating additional holes that enable the dosage form to float and thereby prolonging its longer residence time (Fig. 4). The size and number of these holes were correlated with the 3D structure of the pectin. In the near part of the gel, the holes were smaller in size than in the farther part of the gel (Table 4) [22].

The gradient of the hole size observed within the gel may be linked to the gradient of the mesh-size observed in such anisotropic gels. The near part of the gel was dense and rigid due to the polymer accumulation during the gelation process [23] resulting in small mesh-size (pores). Consequently, when protons from SGF diffused into this part and reacted with dispersed NaHCO₃, the holes will be formed but their size will be restricted due to the high density of the 3D network. In contrast, the farther part of the gel was soft, and the mesh-size was larger, allowing the formation of larger holes due to the reaction of NaHCO₃ and the SGF.

Beside the prolongation of the residence time in SGF, these dosage forms had high encapsulation efficiency (EE) ranged from 84 % to 93 % (Table 1) and the release profile of EGCG is controlled by the erosion and the relaxation of the polymer matrix (Fig. 5). It was then shown that the presence of high amount of NaHCO₃ (GCE_5 %) allowed the formation of large holes and then the release of EGCG was faster compared to (GCE_1 % and GCE_3 %).

## 6. Conclusions

Floating calcium-pectinate hydrogels, incorporating (–)-Epi-gallocatechin-3-gallate (EGCG) as a model drug, were developed through external ionotropic gelation, resulting in two distinct forms: beads or cylinders (Fig. 1). Sodium bicarbonate served as the gas-generating agent, employed at varying concentrations (0, 1 %, 3 %, and 5%w/v). Specifically designed cylindrical gels were cut into three slices (NEAR, MIDDLE, and FAR) to investigate CO₂ gas formation and its distribution within the beads. These investigations revealed the formation of holes in the gel due to the reaction between a small amount of dispersed sodium bicarbonate and protons released during the gelation. A gradient in hole size within the gel was observed, corresponding to the anisotropic nature of the gel. The near part appeared denser and rigid, resulting in smaller holes, while the farther part was softer with larger holes. This unique structure, combined with mucoadhesivity, facilitated buoyancy in gastric fluid, prolonging residence time. The introduction of sodium bicarbonate also affected the pH within the gel, leading to the degradation of EGCG in a pH-dependent manner, specifically in the farthest parts of the gel but not affecting the anti-oxidant properties of EGCG. The dosage forms exhibited high encapsulation efficiency and followed a release controlled by the erosion and the relaxation of the polymer matrix. Notably, higher concentrations of sodium bicarbonate accelerated EGCG release, emphasizing the impact of CO₂ generation on drug release kinetics. Overall, this study highlights the potential of these anisotropic gels as innovative drug delivery systems, particularly for achieving controlled release in the stomach.

### CRediT authorship contribution statement

**Worrawee Siripruekpong:** Writing – review & editing, Writing – original draft, Methodology, Data curation, Conceptualization. **Rue-deekorn Wiwattanapataapee:** Writing – review & editing, Supervision,

<caption>**Table 4**</caption>

Diameter and number of pores in the GCE_5 % dried gel, previously immersed in SGF with a pH of 1.2 for 8 h. Number of pores and surface considered for the calculation per square millimeter (1 mm²) of sample area.

<table><thead><tr><th rowspan="2">Gel formulations</th><th colspan="3">Number of pores (pores/mm²)</th><th colspan="3">Diameter of pore (μm)</th></tr><tr><th>NEAR</th><th>MIDDLE</th><th>FAR</th><th>NEAR</th><th>MIDDLE</th><th>FAR</th></tr></thead><tbody><tr><td>CGE_CaLNa5%</td><td>6000</td><td>340</td><td>11</td><td>11 ± 5</td><td>22 ± 7</td><td>126 ± 40</td></tr></tbody></table>

Resources, Project administration, Funding acquisition. **Odile Cham-bin:** Writing – review & editing, Validation. **Ali Assifaoui:** Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Investigation, Conceptualization.

### Data availability

All data and software supporting the conclusions are provided in this article.

### Consent for publication

All authors have reviewed and approved the final version of this manuscript.

### Ethics approval and consent to participate

The ethics approval was not necessary for this study because it did not include humans or animals.

### Funding

The study was financially supported by the Thailand Research Fund under the Royal Golden Jubilee Ph.D. Program, grant number (PHD/0069/2560).

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

This study was supported by the Thailand Research Fund under the Royal Golden Jubilee Ph.D. Program (PHD/0069/2560). The authors wish to thank Marie-Laure Leonard and Jean-Marc Dachicourt from Polytech Dijon for their technical help and their kind availability for structural analysis (SEM and TGA). The authors also thank the DIVVA technological platform (L'Institut Agro Dijon, Université Bourgogne Europe) for the access to various equipment.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jddst.2025.106729.

### Data availability

Data will be made available on request.

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