Zein/Hyaluronic Acid/Propylene Glycol Alginate Nanoparticle‐Stabilized Pickering Emulsion for the Encapsulation of Astaxanthin as a Delivery System

ABSTRACT

Astaxanthin exhibits significant antioxidant and cardioprotective properties. However, its photodegradability and thermal sensitivity challenge its application. Zein/hyaluronic acid/propylene glycol alginate (zein/HA/PGA) nanoparticles were fabricated using a pH cycling method. The nanoparticles exhibited a contact angle of 89.20 ± 0.32°, indicating favorable amphiphilic characteristics. Fourier‐transform infrared spectroscopy revealed intermolecular interactions among zein, HA, and PGA, consistent with hydrogen bonding and electrostatic attractions. Molecular docking results clarified the binding stability of zein with HA. The zein/HA/PGA nanoparticles at a concentration of 2.4 % (w/v) and an oil phase volume fraction of 0.6 facilitated the formation of Pickering emulsions with high stability and robust ionic strength tolerance (up to 500 mM NaCl). Rheological assessments demonstrated a higher interfacial tension and dilatational elasticity. Astaxanthin was encapsulated by Pickering emulsion, and gastrointestinal release simulations showed a bioavailability of 17.57 ± 0.34%. The zein/HA/PGA nanoparticle‐stabilized Pickering emulsions offer a promising platform for applications in nutraceuticals and cosmeceuticals.

Practical Applications

This study provides a novel strategy for the preparation of Pickering emulsions utilizing food‐grade nanoparticles as stabilizers and expands the scope of applications within the food, cosmetic, and pharmaceutical sectors. Furthermore, the natural encapsulated astaxanthin has a potential to be used as a functional ingredient as the release properties in gastrointestinal conditions.

1. Introduction

Astaxanthin (AST) is a lipophilic carotenoid widely present in crustaceans, avian species, salmon, and certain microalgae, exhibiting potent antioxidant, anti‐aging, anti‐inflammatory, and immunomodulatory properties (Cao et al. 2023). However, AST is highly susceptible to degradation during storage and processing due to factors such as heat, strong acids, and alkalis, which significantly limit its application (Chen et al. 2021). Therefore, numerous delivery systems, including oil gels, liposomes, Pickering emulsions, and protein nanoparticles, have been employed to mitigate the degradation and enhance the aqueous solubility and biological activity of astaxanthin (Zhu et al. 2025). Among these, Pickering emulsions demonstrate outstanding stability by effectively preventing particle aggregation, making them a prominent encapsulation strategy for lipophilic bioactive compounds in nutraceutical and pharmaceutical applications (W. Zhang et al. 2021).

Zein exhibits distinct hydrophobic and hydrophilic surface properties and can self‐assemble to nanoparticles, which have been extensively studied as stabilizers to stabilize Pickering emulsions for the delivery of active ingredients (Ahranjani et al. 2025). However, zein predominantly comprises over 50% non‐polar amino acid residues, with a reduced proportion of basic and acidic amino acids, leading to pronounced hydrophobic characteristics that constrain its utility within the industry. Consequently, it is imperative to modify zein or integrate it with hydrophilic polymers to engineer nanoparticles aimed at augmenting surface hydrophilicity. Fortunately, the amphiphilic character of zein can be modulated by altering the ionization state of its amino acid residues through pH adjustments, enabling its broad application across diverse product formulations. Zein, tannic acid (TA), and sodium alginate (SA) were employed to synergistically improve the stability of Pickering emulsions, as demonstrated by X. Liu et al. (2023). The zein/TA/SA nanoparticles stabilize Pickering emulsions with excellent stability across a range of pH conditions and ionic strengths. Other biopolymers are also commonly utilized in conjunction with zein, such as whey protein isolate, tea saponin, and sophorolipid, which can be dissolved alongside zein in strongly alkaline aqueous solutions (Giteru et al. 2021). Hyaluronic acid (HA) is non‐toxic, biocompatible, and biodegradable and exhibits notable biological activities such as antioxidant and anti‐inflammatory effects (W. Zhang, Huan, et al. 2024). Moreover, owing to its high hydroxyl content, HA can interact with biological macromolecules via non‐covalent interactions and thereby modulate surface hydrophobicity (Salih et al. 2024). Furthermore, propylene glycol alginate (PGA), characterized by its highly charged linear polymer structure, effectively mitigates droplet aggregation through long‐range electrostatic repulsion (X. Zhang et al. 2022).

Therefore, the objective of this work was to prepare zein/HA/PGA nanoparticle using zein, HA, and PGA as stabilizers through a pH‐cycle method. AST was selected as a model compound encapsulating in the Pickering emulsions in this study. The physicochemical properties of the nanoparticles were evaluated based on particle size, polydispersity index (PDI), and zeta potential. The morphology and structure of the obtained nanoparticles were analyzed. The stability and encapsulation properties of the Pickering emulsions were measured. It is anticipated that this study will develop a novel edible nanoparticle and stabilized Pickering emulsions that could be used in foods and pharmaceutical products.

2. Materials and Methods

2.1. Materials

Zein (>98%, molecular weight: 25–45 kDa) was obtained from Shanghai Xingwei Biotechnology Co., Ltd. (Shanghai, China). HA (>98%, molecular weight: 100 kDa) was provided by Shaanxi Wanzhi Source Biotechnology Co., Ltd. (Xi'an, China). Propylene glycol alginate (>98%, viscosity 300 mPa^−s) was obtained from Shaanxi Wanzhi Source Biotechnology Co., Ltd. AST (5.0 % w/v) was supplied by Kunming Baiou Microalgae Technology Co., Ltd. Soybean oil was purchased from a local supermarket. Trypsin (100,000 U/g) and pepsin (10,000 U/g) were provided by Tianjin Huasheng Chemical Reagents Co., Ltd. Lipase (100,000 U/g) was supplied by Shanghai Shifeng Biotechnology Co., Ltd. (Shanghai, China). Nile Red (>98%) and fluorescein isothiocyanate (FITC, >98%) were obtained from Tianjin Baimo Technology Co., Ltd. (Tianjin, China). All other reagents were of analytical grade and purchased from China National Pharmaceutical Group Chemical Reagents Co., Ltd. (Shanghai, China).

2.2. Preparation of Zein/HA/PGA Nanoparticles

The zein/HA/PGA nanoparticles were prepared using a pH‐cycle method (Sun et al. 2018a) with some modifications. Typically, a specified amount of zein was mixed with 100 mL deionized water, with the pH adjusted to 11.5 using 1.0 M NaOH, and was magnetically stirred at 1000 rpm for 30 min until complete dissolution. Notably, during pH adjustment, 1.0 M NaOH was introduced dropwise into the zein aqueous solution via syringe, and the solution was maintained at the target pH for 5 min. An appropriate amount of HA was then added to the alkaline zein solution, and stirring continued at 1000 rpm for another 30 min to achieve complete solubilization. Subsequently, a designated amount of PGA was introduced, and the mixture was stirred magnetically at 1000 rpm for 30 min before being left to stand overnight, resulting in a ternary zein/HA/PGA nanoparticle colloidal solution. A control sample of zein/HA nanoparticles without PGA was also prepared for comparison.

2.2.1. The Effect of Zein:HA on the Properties of Zein/HA/PGA Nanoparticles

Zein (0.8 g) was added to 100 mL of deionized water and mixed; then, the pH was adjusted to 11.5 using the previous method (Section 2.2). The mixture was magnetically stirred at 1000 rpm for 30 min until no visible particles remained. Subsequently, 0.2 g of HA was added, and the mixture was stirred magnetically at 1000 rpm for another 30 min. Then, 0.2 g of PGA was incorporated, followed by a further 30‐min magnetic stirring at 1000 rpm. The mass ratio of zein to HA was maintained at 8:2. Similarly, colloidal solutions with zein:HA ratios of 7:3, 6:4, 5:5, 4:6, 3:7, and 2:8 were prepared, with the PGA concentration fixed at 0.2 % w/v.

2.2.2. The Effect of PGA on Zein/HA/PGA Nanoparticles

Zein (0.7 g) was added to 100 mL of deionized water and mixed thoroughly. The pH was adjusted to 11.5 using the previous method (Section 2.2). Subsequently, 0.3 g of HA was added, and the mixture was stirred magnetically at 1000 rpm for another 30 min to maintain a zein:HA mass ratio of 7:3. Based on this, varying amounts of PGA (0, 0.1, 0.2, 0.3, 0.4, 0.5 g) were sequentially incorporated to investigate the influence of different PGA concentrations on the colloidal stability and properties.

2.3. Characterization of Nanoparticles

2.3.1. Determination of Particle Size and ζ Potential of Zein/HA/PGA Nanoparticles

Zein/HA and zein/HA/PGA nanoparticles were analyzed for nanoparticle size, zeta potential, and PDI using a nanoparticle size and zeta potential analyzer (Zeta100, Malvern) under the condition with dispersant refractive index as 1.33, the viscosity as 0.8872 mPa^−s and signal intensity (∼3700 counts per second). The samples were diluted to an appropriate concentration (1:100) with deionized water. All measurements were conducted at 25°C.

2.3.2. Fourier Transform Infrared Spectra

The interactions among zein, HA, and PGA were evaluated using a Fourier Transform Infrared Spectrometer (Vertex 70, Bruker, Germany). The samples (zein, HA, zein/HA, zein/HA/PGA nanoparticles) were mixed with KBr at a mass ratio of 1:100, ground, and pressed into pellets. Spectra were recorded within the wavenumber range of 4000–400 cm⁻¹ at a resolution of 2 cm⁻¹. To assess repeatability, a total of 128 scans per sample were acquired. The data were assessed using the Omnic software (OMNICTM Series; Thermo Scientific, USA) once all the spectra had been collected.

2.3.3. Wettability Measurement of Nanoparticles

The oil‐in‐water contact angle (θ_O/W) of the zein, HA, and zein/HA/PGA nanoparticles were determined using an automatic surface tension meter (IL4201; Sitar, Germany) to evaluate their hydrophilic and hydrophobic properties. Each freeze‐dried sample was compressed into cylindrical tablets measuring 13 mm in diameter and 2 mm in thickness. Typically, the tablet was placed in an optical glass scraper containing soybean oil to achieve balance for a duration of 8 min. Following that, deionized water (4 µL) was slowly added to the tablet surface using a high‐precision syringe system. Digital images of the resulting droplets were captured via a camera connected to the device. The contact angle (θ_O/W) was determined by simulating the Laplace–Young equation, with three measurements averaged for accuracy.

2.4. Molecular Docking

The zein structure, which is not available in the Protein Data Bank (RCSB), was predicted using AlphaFold 3 (http://alphafoldserver.com/) by the amino acid sequence of zein (ID: Q41844, residues 22–265) obtained from the UniProt database (C. Liu et al. 2021). HA is a linear macromolecular mucopolysaccharide, composed of d‐glucuronic acid and N‐acetyl‐d‐glucosamine that are connected by β‐1, 3 and β‐1, 4 glycosidic bonds, both d‐glucuronic acid and N‐acetyl‐d‐glucosamine were used for molecular docking (Zhang et al. 2024). The 2D structures of these molecules were generated using ChemDraw 22.0.0 and energy‐minimized using the “prepare ligand” program in Discovery Studio (DS). The grid parameters for docking at the active site region of zein were set as follows: center_x = −5.15, center_y = 6.06, and center_z = −4.83; the grid box dimensions were set to size_x = 78.96, size_y = 78.96, and size_z = 78.96. After docking, the conformation with the best binding energy and the highest number of repeated binding was selected as the output result. The resulting conformations were then imported into PyMol 3.1 and Discovery Studio 2019 for visual analysis.

2.5. Preparation of Pickering Emulsion

A specified volume of zein/HA/PGA nanoparticles was employed as stabilizers to formulate oil‐in‐water (O/W) Pickering emulsions. The process involved high‐shear homogenization at 10,000 rpm for 3 min. Subsequently, a predetermined volume of soybean oil was gradually added to the nanoparticle dispersion over 2 min, with the total emulsion volume fixed at 10 mL. To investigate the effect of oil phase volume fraction on emulsion properties, the concentration of zein/HA/PGA nanoparticles was maintained at 2.4 % w/v, while preparing emulsions with varying oil volume fractions (φ = 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7). To assess the influence of nanoparticle concentration, emulsions with a fixed oil phase volume fraction (φ = 0.6) were prepared using different nanocolloid concentrations (0.6%, 1.2%, 1.8%, 2.4%, 3.0%, and 3.6% w/v).

2.6. Characterization of Pickering Emulsions

2.6.1. Microstructure of Pickering Emulsion

The morphological features of the Pickering emulsion were observed using an optical microscope (80i, Nikon, Japan). Additionally, the microstructural interface of the Pickering emulsions was examined with a confocal laser scanning microscope (LSM900; Nikon). In brief, 1.0 mg/mL of fluorescein isothiocyanate isopropanol solvent and 1.0 mg/mL of Nile red isopropanol solvent were prepared. Then, Nile red solution was employed to stain the oil phase, while fluorescein isothiocyanate solution stained the nanocolloids. A 10 µL of the stained emulsion was placed on a glass slide, covered with a coverslip, and observed under a laser confocal microscope. The excitation wavelengths used for Nile red and fluorescein isothiocyanate were 543 and 488 nm, respectively (Y. Wang et al. 2024).

2.6.2. Rheological Property Measurement

The effect of oil volume fraction and nanoparticles concentration on the flow behavior and viscoelastic properties of Pickering emulsions was investigated. The measurements were conducted by a DWS Research Lab apparatus (LS Instruments Inc., Fribourg, Switzerland) equipped with a red laser source (λ = 685 nm, 45 mW). Microrheological properties were investigated using transmission mode. The acquisition duration was set to 30 s, followed by a 30‐s echo duration (Niu et al. 2022). All measurements were conducted at 25°C. Quartz cuvette with an optical path length of 5 mm was employed. Prior to measurement, all samples were equilibrated in the measurement chamber for 5 min. Data processing was conducted using the software provided by LS Instruments Inc. The elastic modulus (G′) and loss modulus (G″) were recorded versus frequency.

2.7. Stability Performance of Pickering Emulsion

2.7.1. Storage Stability

A 10‐mL of freshly treated emulsion was dispensed into centrifuge tube and the storage stability was assessed by standing at room temperature after regular storage periods (1, 7, 14, 21, and 28 days) were recorded. The visual characteristics of the prepared emulsions at different storage periods were also summarized.

The Creaming index (CI) was calculated using the following formula:

$$\mathrm{CI}\%=\left({\mathrm{H}}_1/{\mathrm{H}}_0\right)\times 100\%$$ (1)

where H1 (cm) represented the serum layer, and H0 (cm) was the total height of the emulsion system.

2.7.2. Centrifugal Stability

A 20‐mL sample of Pickering emulsion was placed in a centrifuge tube and subjected to centrifugation at 8000 rpm for 10 min. The oil and stratification on the surface were then observed and recorded.

2.7.3. The Stability of Salt Ions in Pickering Emulsion

Pickering emulsions were prepared using zein/HA/PGA nanoparticles (2.4 % w/v) and soybean oil phase (φ = 0.6), with a total volume of 10 mL. Different concentrations of NaCl solutions (0, 100, 200, 300, 400, and 500 mmol/L) were added. The emulsions were stirred at 10,000 rpm for 3 min.

The appearance and morphology of the emulsion were documented using a digital camera, while its microstructure was examined with an upright fluorescence microscope (80i, Nikon).

2.8. Delivery Systems of Bioactive Components for in Vitro Digestion

To investigate the FFAs and lipophilic bioactive components released from the nanoparticle‐stabilized Pickering emulsion. AST, a representative lipophilic bioactive compound, was incorporated at a concentration of 0.2 µg/mL into the oil phase. Pickering emulsions were stabilized by either zein/HA nanoparticle (mass ratio 7:3, 2.4% w/v) or zein/HA/PGA nanoparticles (mass ratio 7:3:2, 2.4% w/v). A control group utilized bulk oil. These Pickering emulsions were prepared by homogenizing the mixture of soybean oil (60%, v/v) and nanoparticle solution (40%, v/v).

The dynamic in vitro digestion procedure with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was conducted with minor modifications to previously described methods (Tan et al. 2017).

2.8.1. Simulated Gastric Digestion

A freshly SGF was prepared comprising 0.2% (w/v) NaCl, 0.32 % (w/v) pepsin, and 0.7 % (v/v) concentrated HCl, with pH adjusted to 1.2 using 0.5 M HCl. To initiate gastric digestion, 0.5 g of test sample (either Pickering emulsion or bulk oil) as the internal phase was first mixed with 12 mL of phosphate buffer and incubated at 37°C while being magnetically stirred for 10 min. Then, the 12 mL of SGF was added, and the pH of the mixture was adjusted to 2.0 by adding 0.5 M HCl. The mixture was maintained at 37°C while being continuously shaken at 100 rpm for 2 h to mimic the conditions in the human stomach.

2.8.2. Simulated Small Intestinal Digestion

The SIF was prepared containing 39 mmol/L K₂HPO₄, 150 mmol/L NaCl, 30 mmol/L CaCl₂, 0.8% (w/v) bile salts, and 3% (w/v) pancreatin, and the pH was adjusted to pH 7.0 with phosphate buffer. After the digestion in stimulated SGF, the pH of the mixture was adjusted to 7.0 with 0.5 M NaOH to mimic digestion in the small intestine. Then, the same volume of SIF was added. During the 2‐h lipolysis, the pH was maintained at 7.0 by manually adding 0.25 M NaOH. The amount of NaOH consumed over time was recorded throughout the intestinal digestion. Therefore, the percentage of FFAs released was calculated using Equation (2).

$$\mathrm{FFA}(\%)=\frac{{\mathrm{C}}_{\mathrm{NaOH}}\times {\mathrm{V}}_{\mathrm{NaOH}}\times {\mathrm{M}}_{\mathrm{Lipid}}}{2\times {\mathrm{m}}_{\mathrm{Lipid}}},$$ (2)

C_NaOH denotes the molarity of the NaOH solution, V_NaOH represents the volume of NaOH used in the intestinal digestion, M_Lipid indicates the average molecular weight of the oil, and m_Lipid corresponds to the total mass of the oil.

Assessment of AST bioavailability involved centrifuging the digested Pickering emulsion from the intestinal phase at 15,000 g for 30 min at 4°C, followed by collection of the intermicellar phase. AST extraction was performed using a modified version of the previously reported method (X. Liu et al. 2023). Note that 1.0 mL of the aqueous fraction containing formulated AST micelles was mixed with 6.0 mL of an organic solvent mixture (n‐hexane: ethanol = 2:1, v/v) via vortexing and allowed to stand for 2 min. The supernatant was collected and subjected to three successive extractions with the organic solvent. The combined supernatants were analyzed spectrophotometrically at 476 nm. AST concentration was then calculated using the standard curve y = 0.0163x − 0.0152 (R ² = 0.9985, where x represents the AST concentration in µg/L and y represents the corresponding absorbance). The bioavailability of AST was determined using Equation (3):

$$\mathrm{Bioaccessibility}\left(\%\right)=\frac{{\mathrm{C}}_{\mathrm{micelles}}}{{\mathrm{C}}_{\mathrm{original}\mathrm{emulsion}}}\times 100,$$ (3)

where C_micelles represents the concentration of AST within the micelles, while C_original emulsion denotes the concentration of AST in the unprocessed emulsion.

2.9. Statistical Analysis

Each experimental procedure was conducted in triplicate, with all data expressed as mean ± standard deviation. Statistical comparisons between groups were performed using SPSS (version 29.0).

3. Results and Discussion

3.1. Effect of Zein:HA Ratio on Nanoparticles

The dispersibility, size, and charge characteristics of nanoparticles are closely correlated with the stability of emulsion. The impact of diverse zein:HA mass ratios on the mean particle size, PDI, and zeta potential of the nanoparticles was systematically investigated. As illustrated in Figure 1A, when the zein to HA mass ratio varied from 8:2 to 6:4, the mean diameter of the zein/HA/PGA nanoparticles was markedly decreased compared to zein/HA nanoparticles. The integration of HA contributed to the formation of smaller, more densely packed nanostructures, consistent with the findings reported by Zhang et al. (2024). When the zein:HA mass ratio ranges from 5:5 to 2:8, an elevation in HA concentration is associated with a marginal increase in the mean nanoparticle diameter, which remains below 650 nm. As the HA content proportion increases, the PDI values of the nanoparticle exhibit a marked elevation. However, within the ratio range of 8:2 to 7:3, the PDI values remain comparatively low, indicating narrower particle size distribution. Additionally, the surface zeta potential of the nanoparticles critically impacts their colloidal stability. Typically, higher absolute zeta potential values correspond to stronger electrostatic repulsion among nanoparticles, thereby mitigating agglomeration and improving colloidal stability (Ge et al. 2022). Figure 1B demonstrates that a zein:HA mass ratio between 8:2 and 7:3 results in increased measured zeta potential values, signifying improved colloidal stability of the nanoparticles within this specific ratio range. The zein/HA/PGA nanoparticles formulated with a zein:HA mass ratio of 7:3 exhibited enhanced stability in Pickering emulsions, as determined by nanoparticle size distribution and zeta potential analyses. The zein:HA mass ratio of 7:3 was therefore selected for subsequent zein/HA/PGA nanoparticle preparations.

FIGURE 1.

Effect of zein/HA mass ratio on (A) particle size and polydispersity indexes (PDIs) and (B) zeta potential.

3.2. Effect of PGA on Nanoparticles

Figure 2 depicts the effect of different PGA concentrations (0.1%, 0.2%, 0.3%, 0.4%, and 0.5% w/v) on nanoparticle diameter, PDI, and zeta potential measurements. As the PGA concentration increases, the pH of the nanoparticle dispersion gradually decreases. As illustrated in Figure 2A, when the concentrations of PGA reach 0.1% or 0.2% in the zein/HA/PGA nanoparticle colloidal solution, the particle size distribution of the zein/HA/PGA nanoparticles is reduced compared to that of the zein/HA nanoparticles. This suggests that PGA incorporation enhances the intermolecular interactions between zein and HA, thereby promoting a more compact nanoparticle architecture, in accordance with the findings reported by Yu et al. (2023). Figure 2B demonstrates that the zeta potential of zein/HA/PGA nanoparticle exceeds that of zein/HA nanoparticles. These findings indicate that the integration of PGA enhances the stability of zein/HA/PGA nanoparticles compared to zein/HA nanoparticles. Furthermore, all nanoparticles adding PGA exhibited relatively low PDI values (<0.3), suggesting a uniform particle size distribution. As shown in Figure 2A, particle size exhibited a gradual increase with PGA concentrations of 0.3%, 0.4%, and 0.5%. Conversely, when the PGA addition is 0.2%, the nanoparticle showed a more homogeneous size distribution (lower PDI) and improved surface charge characteristics, which are conducive to emulsion stability. Consequently, a PGA concentration of 0.2% was selected for the preparation of zein/HA/PGA nanoparticles at the following research.

FIGURE 2.

Effect of different PGA (A) particle size and polydispersity indexes (PDIs) and (B) zeta potential.

3.3. Characterization of Nanoparticles

3.3.1. Fourier Transform Infrared Spectrometer

The intermolecular interaction can be inferred from the changes in the positions and intensities of the characteristic peaks in Fourier transform infrared (FT‐IR). As shown in Figure 3, the characteristic peaks of zein predominantly appear at 3431, 2957, 1649, and 1507 cm⁻¹, corresponding respectively to the O‐H stretching vibration, methyl C‐H stretching vibration, amide I band (1600–1700 cm⁻¹) involving C = O stretching vibration, and amide II band (1500–1600 cm⁻¹) involving C‐N bending and N‐H bending vibrations. The zein/HA/PGA nanoparticles exhibit a strong peak at 3472 cm⁻¹, shifted to lower wavenumbers compared to zein/HA (peak at 3489 cm⁻¹), indicating the formation of hydrogen bonds during the assembly of the zein/HA/PGA nanoparticles (Q. Liu, Tang, et al. 2025). Distinct peaks at 1649 cm⁻¹ for zein are attributable to C = O stretching vibrations (such as in the amide I band) or amide group vibrations. After PGA was added, there was no significant change of amide II of zein but a blue shift of the amide I band appeared, from 1667 to 1679 cm⁻¹. The results indicated that the hydrogen bonds or hydrophobic effects between zein and PGA may exist, resulting in shifts in vibrational frequencies. These findings suggest that HA and PGA molecules may interact with zein molecules via encapsulation mechanisms, leading to densely structured zein/HA/PGA nanoparticles. Such results are consistent with previous studies by Ye et al. (2023).

FIGURE 3.

FT‐IR spectra of zein, HA, PGA, zein/HA, and zein/HA/PGA.

3.3.2. Surface Wettability

The surface wettability of particles is a critical factor in determining their ability to stabilize emulsions. Figure 4 illustrates the contact angles (θ) of the nanoparticles prepared with different zein:HA mass ratios when PGA is constant. The contact angle decreases as the amount of HA increases, which can be attributed to the hydrophilicity of HA (Salih et al. 2024). When the zein:HA mass ratio is 7:3, the contact angle of the formed nanoparticles is 89.20 ± 0.32°, demonstrating amphiphilic properties with high hydrophilic and lipophilic characteristics. The prepared nanoparticles can be well stabilized at the oil/water interface, playing a significant role in stabilizing Pickering emulsions.

FIGURE 4.

Contact angles of nanoparticles formed using different zein/HA weight ratios measured by dropping a water droplet onto films of the respective nanoparticles in air environment.

3.4. Molecular Docking

Molecular docking was employed to gain insights into the interaction between zein and HA, shedding light on their binding process (Liu et al. 2025). Molecular docking results revealed the presence of hydrogen bonding and van der Waals force interactions between zein and N‐acetyl‐d‐glucosamine/d‐glucuronic acid groups (Figure 5A–F). To be specific, Gln236 and Ser189 of zein were found to form hydrogen bonds with N‐acetyl‐d‐glucosamine groups. Leu232, Phe225, Pro221, Tyr222, Val186, Leu193, and Ala190 of zein were discovered to interact with N‐acetyl‐d‐glucosamine groups in a manner of hydrophobic interaction. Gln176 and SGln177 of zein were found to form hydrogen bonds with d‐glucuronic acid groups. Pro180, Val105, Gln109, Gln108, Leu112, and Ala173 of zein were discovered to interact with d‐glucuronic acid groups in a manner of hydrophobic interaction. In addition, the binding free energy of the N‐acetyl‐d‐glucosamine‐zein complex (−4.2 kcal/mol) is lower than that of the d‐glucuronic acid‐zein complex (−3.6 kcal/mol), suggesting a stronger interaction between N‐acetyl glucosamine and zein. This finding corroborated the secondary structure inference, indicating that the integration of localized HA structure could modulate zein conformation and thus improve the stability of the Pickering emulsion.

FIGURE 5.

(A) Zein‐N‐acetyl‐d‐glucosamine complex (B). Residues on zein interact with N‐acetyl‐d‐glucosamine groups (C). 2D diagrams of zein‐N‐acetyl‐d‐glucosamine interaction (D). Zein‐d‐glucuronic acid complex (E). Residues on zein interact with d‐glucuronic acid groups (F). 2D diagrams of zein‐d‐glucuronic acid interaction.

3.5. Production and Characterization of Pickering Emulsions

3.5.1. Effect of Nanoparticles Concentration on Morphology and Stability

To evaluate the effect of nanoparticle concentration on emulsion stability, Pickering emulsions were prepared by fixing the oil volume fraction (φ = 0.6) and using zein/HA/PGA (7:3:2) nanoparticle solutions with concentrations of 0.6%, 1.2%, 1.8%, 2.4%, 3.0%, and 3.6% (w/v) as continuous phase. Figure 6 illustrates microscopic characterization of Pickering emulsions across a range of nanoparticle concentrations. The results demonstrate that at nanoparticle concentration between 0.6% and 1.8% (w/v), droplet diameters are comparatively larger and exhibit reduced monodispersity. When the nanoparticle concentration reaches 2.4% (w/v) and higher, the droplet distribution becomes increasingly dense with rising nanoparticle concentration, and the droplet sizes are more uniform. Additionally, the average droplet size decreases as the nanoparticle concentration increases, consistent with the findings by Feng et al. (2020) in previous studies. As the nanoparticle concentration escalates, a saturated adsorption monolayer develops at the oil–water interface. Simultaneously, the nanoparticles were uniformly dispersed within the continuous phase to establish a percolating network (Xie et al. 2020), which effectively suppresses the coalescence of dispersed oil droplets, thereby markedly improving the stability of the Pickering emulsion.

FIGURE 6.

Optical microscope images of Pickering emulsions prepared with different zein/HA/PGA 7:3:2 nanoparticles concentrations (0.6%, 1.2%, 1.8%, 2.4％, and 2% w/v) and a fixed oil volume fraction (φ = 0.6).

3.5.2. Effect of the Oil Volume Fraction on Morphology and Stability

To investigate the effect of oil volume fraction on emulsion stability, Pickering emulsions were prepared under different conditions using fixed nanoparticles at a concentration of 2.4% w/v (zein:HA:PGA = 7:3:2) across various oil volume fractions (φ of 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7). The microstructural morphology of the Pickering emulsions was conducted via optical microscopy, with the results depicted in Figure 7. As anticipated, increasing the oil phase volume fraction φ from 0.2 to 0.7 induces a transition in droplet size distribution from a heterogeneous to a more homogeneous and monodisperse system. Additionally, higher oil phase volume increases the density of droplets, which increases the possibility of droplets compression. Consequently, the increase in oil phase volume fraction promotes the viscosity of the emulsion. These phenomena are similar to the observed trends in emulsion structural changes resulting from the stabilization of Pickering emulsions using ethyl cellulose and the alteration of the oil phase volume fraction (Bizmark et al. 2020).

FIGURE 7.

Optical microscope images of Pickering emulsions prepared with different oil volume fractions and a fixed concentration of zein/HA/PGA 7:3:2 nanoparticles (2.4% w/v).

3.6. Microstructure of Pickering Emulsions

Confocal laser scanning microscopy (CLSM) was used to examine the interfacial structure and to confirm that the morphology is critical to their performance. CLSM images (Figure 8) demonstrate that nanoparticles form a distinct interfacial layer at the oil droplet boundary, with green and red fluorescence regions representing the nanoparticles colloid and oil phase, respectively. Additionally, as observed in Figure 8, at an oil volume fraction of 0.3 (Figure 8A), the microstructure of the Pickering emulsion indicates homogeneous oil dispersion, which prevents droplet aggregation and coalescence. Conversely, increasing the oil volume fraction to 0.4 (Figure 8B) makes the distance between adjacent drops gradually narrow. Zein/HA/PGA nanoparticles located on the surface of different droplets entangled with each other, making the relative position of adjacent drops binding to each other and hard to move, thereby enhancing the stability of the Pickering emulsion. A similar behavior was observed in oil‐in‐water (O/W) emulsions stabilized with Panax Notoginseng nanoparticles, which remained phase‐inverted across a series of oil fractions (0.4, 0.5, 0.6, 0.7) (Li et al. 2023).

FIGURE 8.

Confocal laser scanning microscopy images of formulations prepared with a fixed concentration of zein/HA/PGA 7:3:3 nanoparticles dispersion (2.4% w/v) and an oil volume fraction of (A) φ = 0.3; and (B) φ = 0.4. The nanoparticle colloid appears in green (on the left), whereas the emulsion oil phase appears in red (on the right). The images in the middle are an overlay of these two images (right and left).

3.7. Rheological Properties

Shear rheological analysis was employed to elucidate the influence of nanoparticle concentration and oil phase fraction on the viscoelastic behavior of Pickering emulsions. Pickering emulsions were prepared using zein/HA/PGA nanoparticles with a concentration of 2.4 w/v % (7:3:2 ratio) as the continuous phase. The effects of oil volume fractions φ of 0.2, 0.5, and 0.7 on the rheological properties of the emulsions were systematically investigated. The results in Figure 9A show that the apparent viscosity of Pickering emulsions with different φ decreases with the increase of shear rate, exhibiting shear thinning characteristics, indicating that the prepared emulsions belong to the non‐Newtonian fluid behavior. As the φ increases, the apparent viscosity of the Pickering emulsions also rises, attributable to the higher number of droplets, which reduces the aggregation among droplets, thereby elevating the apparent viscosity of Pickering emulsion (X. Zhang et al. 2020). The storage modulus (G′) quantifies the elastic behavior of the Pickering emulsion, whereas the loss modulus (G″) characterizes the viscous response and energy dissipation of Pickering emulsion (Sharkawy et al. 2019). The influence of φ addition on the G′ and G″ of the Pickering emulsion was also measured as a function of frequency (Figure 9B). The G′ exceeds the G′′, indicating that Pickering emulsions exhibit elastic gel network structures. An increase in the φ results in a higher droplet concentration, preventing droplet aggregation and the formation of a cohesive elastic network, thereby conferring the emulsion with characteristic gel‐like rheological behavior.

FIGURE 9.

Effect of oil volume fraction on rheological properties: (A) apparent viscosity versus shear rate, (B) oscillatory frequency sweep curves. G′: storage modulus; G′′: loss modulus.

The effect of nanoparticle concentration on the rheological properties of Pickering emulsions was also investigated using nanoparticles zein/HA/PGA (zein:HA:PGA mass ratio for 7:3:2) at concentrations of 1.8%, 2.4%, and 3.0 w/v % as stabilizers. The φ of oil was maintained at a constant 0.6. As depicted in Figure 10A, an increase in zein/HA/PGA nanoparticles concentration resulted in a corresponding rise in emulsion viscosity. Shear rate escalation led to a reduction in the viscosity of all samples, demonstrating shear‐thinning behavior characteristic of non‐Newtonian fluids. The apparent viscosity of stable Pickering emulsions with varying zein/HA/PGA nanoparticle concentrations decreased progressively with increasing shear rate, confirming shear‐thinning properties. This indicates that the prepared Pickering emulsions are non‐Newtonian fluids, and higher emulsion concentrations correlate with increased viscosity. Similar behavior has been reported in studies utilizing other Pickering stabilizers, such as gliadin/sodium caseinate (Xu et al. 2021). Figure 10B illustrates the stress–frequency rheological profiles of Pickering emulsions stabilized with varying concentrations of zein/HA/PGA nanoparticles. The G' consistently surpasses the G'', indicating that the nanoparticles impart predominantly elastic, gel‐like viscoelastic behavior to the emulsions. This may be attributed to the favorable amphiphilicity of the zein/HA/PGA nanoparticles (θ of 89.20 ± 0.32°), which leads to a relatively stable equilibrium at the oil–water interface and the formation of a stable network structure via steric hindrance. This elastic network results from particle–particle interactions that form a dense, three‐dimensional percolated network within the continuous phase, which enhances the stability emulsion (Zhang et al. 2024).

FIGURE 10.

Effect of the concentration of nanoparticles on rheological properties: (A) apparent viscosity versus shear rate; (B) oscillatory frequency sweep curves. G′: storage modulus; G′′: loss modulus.

3.8. Stabilization of Pickering Emulsions

3.8.1. Centrifugal Stability of Pickering Emulsions

As depicted in Figure 11A, freshly prepared Pickering emulsions with varying φ exhibited no phase separation or oiling‐out phenomena. The emulsions were subjected to centrifugation at 8000 rpm for 10 min to evaluate their stability. The results, as depicted in Figure 11B, indicate that none of the six emulsions exhibited phase separation, except at an φ of 0.2, where slight creaming was observed. This phenomenon is attributed to centrifugation‐induced acceleration of the emulsification process, resulting in droplet concentration and the separation of excess aqueous phase, thereby causing creaming. Additionally, with increasing φ, the propensity for creaming decreased, indicating enhanced emulsion stability with higher oil content (M. Zhang, Chen, et al. 2025). The absence of phase separation in the emulsion following centrifugation indicates that the prepared Pickering emulsion exhibits high stability.

FIGURE 11.

Visual appearance of Pickering emulsions prepared with oil volume fractions of 0.2, 0.3, 0.4, 0.5 and 0.6: (A) pre‐centrifugation; (B) after centrifugation.

3.8.2. Effect of Ionic Strength on the Stability of Pickering Emulsions

Ionic strength is a critical parameter for evaluating the stability of emulsion products. During the production and processing of emulsions, environmental ionic strength may fluctuate due to formulation adjustments, dehydration, and pH variations (Y. Wang et al. 2024). Therefore, emulsion must possess a certain degree of ionic strength tolerance. The prepared emulsions were added with different concentrations of NaCl solution, and the appearance of the emulsions is shown in Figure 12A. It was found that the addition of NaCl solution did not change the stability of the emulsions and that the emulsions maintained a high stability. The microstructure, as depicted in Figure 12B, showed that the droplets were relatively homogeneous and did not change the morphology of the emulsion droplets due to the addition of NaCl. This phenomenon may be attributed to the formation of stable nanoparticles through hydrogen bonding interactions among zein, HA, and PGA, which subsequently adsorb at the oil–water interface. FT‐IR and molecular docking results also confirmed a certain interaction and the generation of hydrogen bonds between zein and HA. This adsorption likely prevents the disruption of Pickering emulsions within specific ionic concentration ranges. Concurrently, the zeta potential (Figure 2) indicates that the zein/HA/PGA nanoparticles possess a net surface charge, conferring a degree of ionic repulsion in NaCl solutions, thereby demonstrating excellent ionic strength resistance.

FIGURE 12.

(A) Visual appearance of Pickering emulsions stabilized by 2.4% zein/HA/PGA nanoparticles with the oil volume fraction φ is 0.6 at different ionic strengths. (B) Microscopy images of Pickering emulsions stabilized by zein/HA/PGA nanoparticles under different NaCl solution concentrations.

3.8.3. Effect of Storage Time on the Stability of Pickering Emulsions

The appearance of Pickering emulsions with varying oil phase volume fractions (φ ranging from 0.2 to 0.6) is depicted in Figure 13A. As the φ decreases from 0.6 to 0.2, localized flocculation and aggregation occur within the emulsion, resulting in a gradual increase in phase separation height. The creaming index (CI) of the Pickering emulsions increased from 8% to 30% as the φ decreased from 0.6 to 0.2 (Figure 13B). Although the CI is comparatively elevated at a φ of 0.2, no substantial demulsification processes were observed. These results imply that oil droplets are effectively encapsulated by zein/HA/PGA nanoparticles and that emulsion stability is enhanced with increasing oil phase volume fraction. This stabilization mechanism is likely attributable to the surface charge characteristics and amphiphilic properties of the zein/HA/PGA nanoparticles.

FIGURE 13.

(A) Visual appearance and creaming index % (CI%) values of Pickering emulsions prepared with oil volume fractions of 0.2, 0.3, 0.4, 0.5, and 0.6, and a dispersion of zein/HA /PGA 7:3:2 nanoparticles (2.4% w/v) right after preparation and (B) after 1 month at room temperature (the vertical line indicates separation).

3.9. In Vitro Intestinal Digestion

The FFA release profile of Pickering emulsions is depicted in Figure 14A. The FFA release profiles of all samples exhibit a similar kinetic pattern, characterized by an initial rapid increase followed by a gradual plateau. This is primarily attributed to the accumulation of lipid digestion products at the oil–water interface following emulsion digestion, which inhibits pancreatic lipase from accessing triglycerides (L. Wang et al. 2023). Following the complete gastrointestinal digestion process, the FFA content released from the bulk oil was 18.17 ± 0.29%. The lipid solubilization rate and extent were significantly enhanced in Pickering emulsions stabilized by zein/HA nanoparticles and zein/HA/PGA nanoparticles. The sequence of FFA release rates is as follows: zein/HA/PGA nanoparticles stabilized Pickering emulsion (48.43 ± 0.51%) > zein/HA nanoparticles stabilized Pickering emulsion (38.43 ± 0.40%) > bulk oil (18.17±0.29%). This is primarily due to the smaller droplet size in the zein/HA/PGA nanoparticles stabilized Pickering emulsion, which increases the interfacial contact area between lipid droplets and digestive enzymes, thereby enhancing lipolytic hydrolysis (Ma et al. 2020). The microstructural analysis of oil droplets in Figure 14B was also validated, showing that large oil droplets within the zein/HA nanoparticle‐stabilized Pickering emulsion significantly aggregated after gastric digestion. The ζ potential of the emulsion decreased, indicating that the low pH in the gastric environment promoted droplet coalescence. Conversely, the aggregation of large oil droplets in the zein/HA/PGA nanoparticle‐stabilized Pickering emulsion was scarcely observed. This may be attributed to the incorporation of PGA, which facilitates the formation of a robust interfacial barrier between zein/HA/PGA nanoparticles and gastric proteases and lipid droplets (Sun et al. 2018b). This indicates that bioactive compounds encapsulated within zein/HA/PGA nanoparticles exhibit enhanced stability in the gastric environment. Figure 14C indicates that the zeta potential of the zein/HA/PGA nanoparticle‐stabilized Pickering emulsion (−43.1 mV) is lower than that of the zein/HA nanoparticle‐stabilized Pickering emulsion (−38.4 mV). The high electrostatic repulsion in zein/HA/PGA nanoparticle‐stabilized Pickering emulsions indicates low inter‐droplet attraction and enhanced emulsion stability. Both zein/HA nanoparticle‐stabilized Pickering emulsions and zein/HA/PGA nanoparticle‐stabilized Pickering emulsions exhibit high negative zeta potential, likely attributable to the presence of anionic constituents such as free fatty acids (Salvia‐Trujillo et al. 2013).

FIGURE 14.

(A) FFA release profiles of astaxanthin in emulsions and bulk oil as a function of small intestine digestion time, (B) fluorescence inverted microscopy observed in vitro simulated gastrointestinal digestion, (C) zeta potential of emulsions, and (D) bioaccessibility of astaxanthin in emulsions and bulk oil.

Furthermore, the bioavailability of AST encapsulated within Pickering emulsions was also assessed, as illustrated in Figure 14D. Generally, triglycerides are hydrolyzed by lipases into diacylglycerols, monoacylglycerols, and FFA, which then form micelles through interactions with bile salts and phospholipids. AST dissolved in oil gradually releases into micelles, converting it into biodegradable compounds (Huang et al. 2021). As anticipated, the bioavailability of AST in the zein/HA/PGA nanoparticle‐stabilized Pickering emulsion (17.57 ± 0.34%) is significantly higher than that in bulk oil (4.18 ± 0.10%), indicating that the zein/HA/PGA nanoparticle‐stabilized Pickering emulsion effectively enhances the bioavailability of AST. The underlying mechanisms may include the following: first, the high viscosity and gel‐like network structure of the emulsion extend the diffusion pathways for oxygen and free radicals, thereby inhibiting the oxidative degradation of AST; secondly, zein/HA/PGA nanoparticles effectively adsorb onto the oil droplet surface, forming a robust protective layer that impedes the ingress of oxidants (Yi et al. 2021). It is evident that the Pickering emulsion stable by zein/HA/PGA nanoparticle demonstrates superior capability for delivering AST during gastrointestinal digestion.

4. Conclusion

This study successfully prepared zein/HA/PGA nanoparticles for stabilizing Pickering emulsions and loading AST. The zein/HA/PGA nanoparticles (mass ratio = 7: 3:2) exhibit near‐neutral wettability (water contact angle: 89.20 ± 0.32°) and demonstrate superior stability to Pickering emulsions. Furthermore, increasing the oil phase volume fraction and nanoparticle concentration within the Pickering emulsion effectively suppressed instability. Additionally, these nanoparticles also exhibited shear‐thinning and elastic‐like behavior. Notably, AST‐loaded Pickering emulsions exhibited a controlled release profile and favorable bioavailability. This research presents a novel methodology for the preparation of Pickering emulsions employing food‐grade nanoparticles as delivery systems for bioactive molecules. Future work will focus on in vivo animal studies, including investigations into the intestinal absorption kinetics and pharmacodynamic effects of AST‐loaded Pickering emulsions.

Nomenclature

AST

astaxanthin

CLSM

confocal laser scanning microscopy

FFA

free fatty acid

FT‑IR

Fourier transform infrared spectroscopy

HA

hyaluronic acid

PDI

polydispersity index

PGA

propylene glycol alginate

SA

sodium alginate

SGF

simulated gastric fluid

SIF

simulated intestinal fluid

TA

tannic acid

Author Contributions

Shanfu Wang: investigation, validation, data curation, writing – original draft. Yan Zhao: formal analysis, validation. Fuge Niu: visualization, formal analysis. Huien Zhang: formal analysis. Jiayan Zheng: visualization. Zhongfa Chen: funding acquisition, supervision. Jian Zhang: writing – review and editing, supervision, conceptualization, funding acquisition.

Conflicts of Interest

The authors declare no conflicts of interest.