Zein–Pectin Nanoparticles for Luteolin Delivery: Physicochemical Characterization, Antioxidant Activity, and Simulated Gastrointestinal Behavior

ABSTRACT

Luteolin (LUT) is a hydrophobic flavonoid with relevant antioxidant potential; however, its application in food and nutraceutical systems is limited by poor aqueous solubility, low dispersibility, and restricted gastrointestinal availability. In this study, luteolin‐loaded zein–pectin nanoparticles (ZPEC/LUT NP) were developed by antisolvent precipitation using pectin as the sole stabilizing polysaccharide. ZPEC/LUT NP showed a mean diameter of 345 ± 4 nm, polydispersity index (PDI) of 0.206 ± 0.019, negative zeta potential (−27± 1 mV), and high encapsulation efficiency (87% ± 11%). The similarity between unloaded and loaded nanoparticles indicated that LUT incorporation did not markedly alter particle size, morphology, or surface charge. Fourier Transform Infrared (FTIR), x‐ray diffraction (XRD), and thermal analyses supported physical encapsulation of LUT within the zein–pectin matrix, with LUT present mainly in an amorphous or molecularly dispersed state. Short‐term colloidal stability studies showed that ZPEC/LUT NP maintained nanometric dimensions and acceptable PDI values during 60 days of storage at room temperature and 10°C. During simulated gastrointestinal digestion, LUT was gradually transferred into soluble fractions, reaching an apparent bioaccessibility of approximately 16% after 6 h. In the ABTS assay, ZPEC/LUT NP exhibited higher apparent radical scavenging activity than free LUT, with IC₅₀ values of approximately 12.5 and 22.5 µg/mL, respectively. Unloaded nanoparticles showed only limited antioxidant contribution. Overall, zein–pectin nanoparticles represent a promising food‐grade platform for improving the physicochemical performance and apparent antioxidant activity of hydrophobic flavonoids, although further studies are required to evaluate long‐term stability and biological performance under physiological conditions.

1. Introduction

Luteolin (3′,4′,5,7‐tetrahydroxyflavone) (LUT) is a naturally occurring flavonoid widely distributed in fruits, vegetables, and medicinal plants (Zhu et al. 2024). It has attracted considerable attention due to its broad spectrum of biological activities, including antioxidant (Ahmadi et al. 2020; Fernando et al. 2024), anti‐inflammatory (Gendrisch et al. 2021; Wang et al. 2024), and anticancer (Hussain et al. 2021; Imran et al. 2019; Zhang and Ma 2024). In recent years, growing evidence has demonstrated its therapeutic potential in managing chronic disorders associated with oxidative stress, such as neurodegenerative diseases (e.g., Alzheimer's and Parkinson's) (Kwon 2017; Goyal et al. 2024; Vongthip et al. 2024; Chib et al. 2025) and cancer (Zheng et al. 2023; Jiang et al. 2021; Rocchetti et al. 2023). These properties are mainly associated with the presence of multiple hydroxyl groups in its chemical structure, which enable LUT to scavenge reactive oxygen species, chelate metal ions, and modulate signaling pathways involved in oxidative stress and inflammation. For this reason, LUT has been investigated as a promising bioactive compound for functional foods, nutraceuticals, and health‐promoting formulations.

Despite these promising pharmacological properties, the practical application of LUT in food and nutraceutical systems remains significantly limited by its unfavorable physicochemical characteristics. LUT exhibits low aqueous solubility and poor permeability, which result in reduced dissolution in gastrointestinal fluids, which restricts dispersion, absorption, and final biological performance after oral administration. Furthermore, LUT is susceptible to chemical degradation under environmental stresses, such as light, oxygen, and pH variations, which can compromise its stability and biological activity (Wang et al. 2021; Taheri et al. 2021; Hayasaka et al. 2018). These limitations reduce the amount of bioactive compound that remains available during processing, storage, and gastrointestinal transit. Therefore, the development of food‐grade delivery systems able to improve LUT dispersibility, protect it from degradation, and modulate its release is an important strategy to expand its application in functional foods and nutraceutical products.

Biopolymer‐based nanoparticles have emerged as useful carriers for hydrophobic bioactive compounds because they can be produced from renewable, biodegradable, and generally recognized as safe materials (Richards et al. 2024; Hoshyar et al. 2016). Among them, zein, a hydrophobic prolamin obtained from corn, has been widely explored as a nanocarrier‐forming protein due to its amphiphilic structure and self‐assembly behavior in aqueous media. During antisolvent precipitation, the reduction in ethanol concentration decreases zein solubility and promotes the formation of colloidal particles capable of entrapping hydrophobic nutraceuticals within a protein‐rich matrix (Patel and Velikov 2014; Giteru et al. 2021; Huang, Sun, et al. 2023). Zein nanoparticles have been reported to improve the dispersibility, stability, and release profile of several poorly water‐soluble bioactives. However, zein particles alone often display limited colloidal stability, especially under changes in pH, ionic strength, or gastrointestinal conditions, because hydrophobic interactions may favor aggregation and precipitation (Liu et al. 2019; Abdelsalam et al. 2021; Wiggers et al. 2022; Ziebarth et al. 2024). This instability restricts their practical application in food systems.

To overcome this drawback, polysaccharides have been incorporated into zein‐based systems as stabilizing macromolecules. These compounds may adsorb onto the particle surface and provide electrostatic and steric stabilization, reducing aggregation and improving resistance to environmental stresses. Pectin is particularly attractive for this purpose because it is an anionic polysaccharide widely used in food systems, with good biocompatibility, biodegradability, and functional properties related to its hydroxyl and carboxyl groups (Chang et al. 2017a; Khorasani and Shojaosadati 2017; Huang, Yao, et al. 2023; Gu et al. 2024). In zein–pectin nanoparticles (ZPEC NP), pectin may act as an external stabilizing layer, contributing to surface charge, hydration, and colloidal protection. In addition, pectin has been associated with mucoadhesive behavior due to its ability to establish hydrogen bonding and intermolecular interactions with mucosal glycoproteins. Although mucoadhesion was not investigated in the present study, this characteristic may be advantageous for oral delivery systems because prolonged residence at the gastrointestinal interface could potentially favor interaction with the absorptive surface and improve the functional performance of encapsulated bioactive compounds. Furthermore, pectin may contribute to the overall antioxidant properties of the formulation, providing multifunctional characteristics beyond colloidal stabilization (Huang, Sun, et al. 2023; Celli et al. 2020; Khorasani and Shojaosadati 2017; Fin et al. 2025).

Several zein‐based delivery systems have been proposed to improve the dispersion and functional performance of hydrophobic flavonoids, including LUT and structurally related polyphenols. In many of these systems, zein is combined with additional stabilizing components, such as surfactants, lecithin, sodium caseinate, gum arabic, proteins, or multiple polysaccharides (Shinde et al. 2019; Li, Zhang, Jin, et al. 2023; Xu et al. 2023). The use of such multicomponent architectures is generally justified by the intrinsic colloidal instability of zein nanoparticles when used alone. Because zein is rich in hydrophobic amino acid residues, particles formed by antisolvent precipitation may undergo aggregation, especially under changes in pH, ionic strength, temperature, or gastrointestinal conditions. Therefore, auxiliary stabilizers are frequently incorporated to provide electrostatic repulsion, steric protection, or interfacial reinforcement.

Although these multicomponent systems can improve nanoparticle performance, increasing formulation complexity may also introduce additional variables affecting reproducibility, compositional consistency, scalability, and mechanistic interpretation. When several stabilizers are simultaneously present, it becomes difficult to identify the individual contribution of each component to particle formation, colloidal stability, bioactive retention, and antioxidant performance. From a food and nutraceutical formulation perspective, simplified architectures based on a reduced number of food‐grade ingredients may therefore be advantageous.

Pectin was selected in the present study as the sole stabilizing polysaccharide because of its anionic character, food‐grade status, biocompatibility, biodegradability, and ability to form hydrated interfacial layers. Its carboxyl and hydroxyl groups can interact with zein through electrostatic interactions, hydrogen bonding, and other non‐covalent forces, particularly under mildly acidic conditions. When adsorbed onto the surface of zein nanoparticles, pectin can confer negative surface charge and steric protection, thereby reducing particle aggregation and improving colloidal stability (Chang et al. 2017a, 2017b). These characteristics support its use as a standalone stabilizer in zein‐based nanoparticles.

In this context, the present study was designed to evaluate a simplified ZPEC NP system for LUT delivery, using pectin as the only stabilizing polysaccharide. This approach allows a clearer assessment of the ability of pectin to stabilize LUT‐loaded zein nanoparticles and provides insight into the physicochemical behavior, antioxidant performance, and simulated gastrointestinal behavior of a reduced‐complexity food‐grade nanocarrier.

2. Materials and Methods

2.1. Chemicals and Materials

Absolute ethyl alcohol 99.8% (Neon), sodium caseinate (Biotec), dimethyl sulfoxide (DMSO‐Synth), luteolin (Sigma Chemical), methanol (Biotec), pectin (Sigma Chemical), zein (Sigma Chemical), and HPLC‐grade acetonitrile (≥99.9%) were purchased from Sigma‐Aldrich, USA.

2.2. Preparation of Zein/Pectin Nanoparticles Loaded With LUT (ZPEC/LUT NP)

LUT‐loaded zein–pectin nanoparticles (ZPEC/LUT NP) were prepared by a liquid–liquid dispersion technique, adapted from the method described by Li, Zhang, Chen, et al. (2023), with modifications. Initially, a 15 mg/mL zein solution was prepared by dissolving zein in 87% (v/v) ethanol under magnetic stirring for 30 min at room temperature. Simultaneously, a 1% (w/v) pectin solution was prepared in ultrapure water, and the pH was adjusted to 4.5 using 0.1 M HCl or NaOH. LUT (10 mg) was first dissolved in a binary solvent mixture composed of 400 µL of DMSO and 1600 µL of 85% hydroalcoholic solution. From this stock, 250 µL was added dropwise to 2750 µL of the zein solution under magnetic stirring and mixed for 30 min to ensure homogeneous distribution of the LUT within the organic phase. This LUT‐loaded organic phase (zein–LUT mixture) was then added dropwise into 15 mL of the aqueous pectin solution under constant magnetic stirring at 1500 rpm. The mixture was stirred for an additional 30 min to allow complete nanoparticle formation. The resulting suspension was subjected to rotary evaporation at 37°C for 20 min to remove residual organic solvents. Nanoparticles were collected by ultracentrifugation at 14,000 rpm for 15 min at 25°C. The supernatant was collected for determination of encapsulation efficiency, and the nanoparticle pellet was resuspended in ultrapure water for subsequent analyses.

Unloaded ZPEC NP were prepared using the same procedure described above, but without the addition of LUT. In this case, the corresponding volume of solvent mixture without LUT was added to the zein solution before dispersion into the pectin phase. ZPEC NP was used as control formulations for physicochemical characterization, morphology, Fourier transform infrared (FTIR), x‐ray diffraction (XRD), and antioxidant activity analyses.

2.3. Physicochemical Analysis

2.3.1. Average Diameter and Polydispersity Index (PDI)

The average diameter of the nanoparticles was determined using dynamic light scattering (DLS) (BIC 90 Plus, Brookhaven Instruments Corp.). For measurement, nanoparticle suspensions were diluted in purified water, placed in polyethylene cuvettes, and analyzed at 25°C with a 659 nm wavelength. Each sample was analyzed over a 3‐min period, with the mean diameter and standard deviation (SD) calculated from 10 replicates. PDI was assessed by analyzing the autocorrelation function during DLS measurements, which assesses particle size uniformity.

2.3.2. Zeta Potential

Zeta potential was measured on the basis of the electrophoretic mobility of the nanoparticles. For this, nanoparticles were resuspended in a 1 mM potassium chloride (KCl) solution at a dilution ratio of 1:200. The suspension was placed in an electrophoretic cell (Nano ZS90, Malvern Instruments, UK), maintained at 25°C, and analyzed over a range of ±150 mV. All measurements were conducted in replicates, with results reported as mean values with their SDs.

2.3.3. Encapsulation Efficiency

The encapsulation efficiency of LUT was determined indirectly by quantifying the nonencapsulated fraction after nanoparticle separation. Briefly, freshly prepared ZPEC/LUT NP suspensions were centrifuged at 14,000 rpm for 15 min at 25°C. After centrifugation, the supernatant was carefully collected and used for the quantification of nonencapsulated LUT.

To minimize analytical errors associated with the poor aqueous solubility of LUT, the recovered supernatant was diluted with methanol:DMSO (9:1, v/v), vortexed for 2 min, sonicated for 5 min, and filtered through a 0.22 µm membrane before HPLC analysis. This solvent‐assisted preparation step was used to ensure complete solubilization of LUT present in the supernatant prior to chromatographic quantification.

A polymer‐free control was also prepared to evaluate whether nonencapsulated LUT could sediment under the experimental conditions used for nanoparticle preparation and separation. For this control, LUT was dissolved in the same DMSO/hydroalcoholic solvent system used for ZPEC/LUT NP preparation, diluted under the same aqueous conditions, and subjected to the same centrifugation procedure. The LUT content recovered after centrifugation was compared with the initial LUT amount to assess possible spontaneous sedimentation in the absence of zein and pectin.

LUT quantification was performed using a Waters 2695 Alliance HPLC system equipped with a LiChrospher column. The mobile phase consisted of acetonitrile:water (45:55, v/v), delivered at a flow rate of 1 mL/min. The column temperature was maintained at 30°C, the total run time was 5 min, and detection was performed at 357 nm, according to the method adapted from Diedrich et al. (2023).

Encapsulation efficiency was calculated according to the following equation:

$$\mathrm{EE}\left(\%\right)=\frac{L_1-L_2}{L_1}\times 100$$ (1)

where L ₁ is the total amount of LUT initially added to the formulation, and L ₂ is the amount of nonencapsulated LUT quantified in the supernatant after nanoparticle separation and solvent‐assisted sample preparation.

2.3.4. Morphology

The morphological characteristics of the nanoparticles were examined using a scanning electron microscope (SEM, TESCAN). For image acquisition, a small volume of the nanoparticle's suspension was carefully deposited onto a sample holder. Following the drying process, the sample surface was coated with a thin layer of gold. The micrographs were obtained using an accelerating voltage of 30 kV and a magnification of 30,000×.

2.3.5. Fourier Transform Infrared Spectroscopy

FTIR was performed using the attenuated total reflectance (ATR) accessory. Spectral analyses were conducted in the range of 650–4000 cm⁻¹ using a Frontier FTIR spectrophotometer (PerkinElmer).

2.3.6. X‐Ray Diffraction

XRD was performed using a D2 PHASER diffractometer (Bruker), operating at a current of 10 mA and a voltage of 30 kV. Samples were placed on glass holders and scanned over a 2θ range of 5°–60°, with a scanning rate of 1 min⁻¹.

2.3.7. Thermogravimetric Analysis (TGA)

TGA and derivative thermogravimetry (DTG) were performed using an STA 6000 instrument (PerkinElmer). Samples were placed in platinum crucibles and heated from 0°C to 800°C at a heating rate of 10°C/min, under nitrogen atmosphere with a carrier gas flow of 100 mL/min.

2.4. Short‐Term Colloidal Stability Study

The short‐term colloidal stability of ZPEC/LUT NP was evaluated by monitoring changes in mean particle diameter and PDI during storage. Freshly prepared nanoparticle suspensions were transferred to closed glass vials and stored under two temperature conditions: room temperature (∼25°C) and refrigerated conditions (10°C).

Samples were maintained under the selected storage conditions for 60 days and analyzed at predetermined time points as follows: 1, 7, 14, 21, 28, and 60 days. At each time point, aliquots were withdrawn and diluted in purified water prior to analysis. Mean particle diameter and PDI were determined by DLS at 25°C, as described in Section 2.3.1.

All measurements were performed in triplicate, and results were expressed as mean ± SD. Statistical differences among storage times within each temperature condition were evaluated using one‐way analysis of variance (ANOVA) followed by Tukey's post hoc test, considering p < 0.05 as statistically significant.

2.5. In Vitro Simulated Gastrointestinal Digestion and Apparent Bioaccessibility of LUT

An in vitro simulated gastrointestinal digestion assay was performed to evaluate the gastrointestinal behavior and apparent bioaccessibility of LUT encapsulated in ZPEC NPs under sequential gastric and intestinal conditions. The experimental design aimed to monitor the transfer of LUT into soluble fractions during digestion and investigate the behavior of the nanoparticle system under conditions simulating gastrointestinal transit.

Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to a modified static digestion procedure. SGF consisted of NaCl solution containing pepsin under acidic conditions (pH 1.2), whereas SIF consisted of phosphate buffer supplemented with pancreatin and bile salts adjusted to pH 6.8. Digestive enzymes and bile salts were included to mimic gastrointestinal conditions and facilitate matrix degradation and solubilization processes associated with hydrophobic compounds.

Briefly, an aliquot of nanoparticle suspension (0.5 mL) was mixed with 7 mL of SGF and incubated at 37 ± 0.5°C for 2 h under continuous agitation (100 rpm) to simulate gastric digestion. Following incubation, samples were centrifuged at 14,000 rpm for 15 min. This centrifugation step was employed as an analytical fractionation procedure to separate LUT transferred into the soluble phase from LUT remaining associated with nanoparticle or precipitated fractions. The recovered supernatant was collected and analyzed as the soluble fraction obtained after gastric digestion, whereas the precipitated material was recovered for the subsequent intestinal stage.

The precipitated fraction was then resuspended in 7 mL of SIF and incubated at 37°C under continuous agitation (100 rpm) for an additional 4 h. During this phase, digestive enzymes and bile salts promoted gradual matrix disassembly and facilitated the transfer of hydrophobic compounds into soluble colloidal structures. After completion of the intestinal stage, samples were centrifuged again at 14,000 rpm for 15 min, and the resulting supernatants were collected as the soluble fractions obtained after intestinal digestion.

LUT concentration in the recovered fractions was quantified by HPLC at 357 nm. All experiments were performed in triplicate, and results were expressed as mean ± SD (n = 3).

The apparent bioaccessibility of LUT was calculated according to the following equation:

$$\mathrm{Apparent}\mathrm{bioaccessibility}\left(\%\right)=\frac{L_{\mathrm{SGF}}+L_{\mathrm{SIF}}}{L_0}\times 100$$ (2)

where $L_{\mathrm{SGF}}$ is the amount of LUT quantified in the supernatant after the gastric phase, $L_{\mathrm{SIF}}$ is the amount of LUT quantified in the supernatant after the intestinal phase, and $L_0$ is the initial amount of LUT in the formulation before digestion.

Considering the sequential fractionation design employed in this study, the obtained values were interpreted as apparent bioaccessibility, reflecting cumulative LUT transfer into soluble fractions throughout digestion rather than absolute physiological intestinal bioaccessibility.

2.6. Evaluation of Antioxidant Activity

The antioxidant activity of ZPEC/LUT NP was determined using the ABTS•⁺ radical scavenging assay, adapted from the method described by Re et al. (1999), with minor modifications. The ABTS•⁺ cation radical was generated by reacting 10 mmol/L ABTS solution with 2.45 mmol/L potassium persulfate, followed by incubation in the dark at room temperature for 24 h to allow complete radical formation. The resulting ABTS•⁺ solution was then diluted with phosphate‐buffered saline (PBS, pH 7.4) to achieve an absorbance of approximately 0.70 ± 0.02 at 734 nm. Samples of free LUT, ZPEC NP, and ZPEC/LUT NPs were prepared in water at concentrations of 6.25, 12.5, 25, and 50 µg/mL. For the assay, 10 µL of each sample was mixed with 190 µL of the diluted ABTS•⁺ solution in 96‐well microplates and incubated for 30 min at room temperature under continuous stirring (500 rpm). After incubation, absorbance was measured at 734 nm using a microplate spectrophotometer. The antioxidant activity was calculated as the percentage inhibition of the ABTS•⁺ radical according to the following equation:

$$\mathrm{ABTS}\mathrm{inhibition}\left(\%\right)=\frac{A\_\mathrm{control}-A\_\mathrm{sample}}{A\_\mathrm{control}}\times 100$$ (3)

where A_control is the absorbance of the ABTS•⁺ solution without sample, and A_sample is the absorbance after reaction with the test sample. All measurements were performed in triplicate, and results were expressed as mean ± SD.

2.7. Statistical Analysis

All results were expressed as mean ± SD. Experimental data were compared using two‐way ANOVA with Tukey's post hoc test, considering a significance level of 5%.

3. Results and Discussion

3.1. Preparation and Physicochemical Characterization of ZPEC/LUT NP

ZPEC/LUT NPs were successfully prepared using the antisolvent precipitation method employing zein as the nanoparticle‐forming protein and pectin as the stabilizing polysaccharide. A schematic representation of the proposed nanoparticle formation mechanism and structural organization is presented in Figure 1. As illustrated, the rapid diffusion of ethanol into the aqueous phase promotes zein supersaturation and nucleation, followed by self‐assembly processes driven mainly by hydrophobic interactions (Yu et al. 2020). During particle formation, LUT molecules become associated with hydrophobic zein domains, whereas pectin adsorbs onto the external surface, forming a hydrated layer that contributes to colloidal stabilization and negative surface charge.

FIGURE 1.

Schematic illustration of the proposed formation mechanism and structural organization of ZPEC/LUT NP.

Adjustment of the pectin solution to pH 4.5 was essential for obtaining reproducible nanoparticles with adequate colloidal stability. Under these conditions, electrostatic interactions between zein and pectin are favored, whereas the hydrated polysaccharide layer contributes to particle stabilization and reduces aggregation (Veneranda et al. 2018). The optimized formulation containing 1.53% zein and 0.10% pectin produced nanoparticles with suitable physicochemical properties for oral delivery of hydrophobic nutraceuticals.

The physicochemical properties of ZPEC NP and ZPEC/LUT NP are summarized in Table 1. Both formulations presented particle diameters within the nanometric range and low PDI values (<0.3), indicating relatively homogeneous colloidal populations.

TABLE 1.

Physicochemical properties of zein–pectin nanoparticle (ZPEC NP) and ZPEC/LUT NP.

Formulation	Mean diameter (nm)	PDI	Zeta potential (mV)	EE (%)
ZPEC NP	356 ± 10	0.254 ± 0.015	−23 ± 2	—
ZPEC/LUT NP	345 ± 4	0.206 ± 0.019	−27 ± 1	87 ± 11

Note: Values are expressed as mean ± SD (n = 3).

Abbreviation: PDI, polydispersity index.

LUT incorporation did not substantially alter nanoparticle size, with ZPEC NP exhibiting an average diameter of 356 nm and ZPEC/LUT NP showing 344 nm. Similarly, a slight reduction in PDI was observed after LUT loading (0.254–0.206), suggesting that incorporation of LUT did not destabilize the nanoparticle system.

The DLS size distribution profile is presented in Figure 2a. ZPEC/LUT NP exhibited a bimodal distribution profile, with one particle population around 100 nm and a second predominant population between approximately 300 and 400 nm. Although this profile indicates a certain degree of heterogeneity, the PDI value below 0.3 suggests acceptable colloidal uniformity for a zein‐based nanoparticle system prepared by antisolvent precipitation. Such behavior may be associated with self‐assembly dynamics occurring during rapid solvent displacement and particle formation.

FIGURE 2.

(a) Dynamic light scattering (DLS) size distribution profile of ZPEC/LUT NP showing a bimodal particle distribution; (b) scanning electron microscopy (SEM) image ZPEC/LUT NP; and (c) SEM image of ZPEC NP. Scale bars indicated in SEM images. PDI, polydispersity index; ZPEC NP, zein–pectin nanoparticle.

These results are consistent with previous studies on zein‐based delivery systems, where nanoparticles ranging between 300 and 400 nm with moderate PDI values have shown promising outcomes for oral drug delivery, particularly in nutraceutical and prophylactic applications. Nanocarriers within this size range can enhance the gastrointestinal stability and sustained release of bioactive compounds, such as LUT (Li et al. 2023; Penalva et al. 2017; Tivano and Chiono 2023).

SEM images of ZPEC/LUT NP and ZPEC NP are shown in Figure 2b,c, respectively. ZPEC/LUT NP displayed predominantly spherical particles, with representative diameters ranging from approximately 220 to 315 nm. ZPEC NP also showed spherical morphology, although with a greater tendency to form clustered structures after drying. Representative particle diameters for ZPEC NP ranged from approximately 143 to 287 nm. The smaller sizes observed by SEM compared with DLS may be attributed to the dry‐state nature of SEM analysis, whereas DLS measures the hydrodynamic diameter of particles in suspension, including the hydrated pectin layer. Comparison between unloaded and loaded nanoparticles indicated that LUT incorporation did not induce major morphological changes. Both formulations retained spherical morphology and nanometric dimensions, supporting the formation of a stable zein–pectin structure after flavonoid encapsulation.

Both unloaded and loaded nanoparticles exhibited negative zeta potential values, with ZPEC NP presenting −23 mV and ZPEC/LUT NP showing −27 mV (Table 1). The observation that unloaded nanoparticles already displayed a negative surface charge suggests that pectin was the major contributor to nanoparticle electrokinetic behavior. Due to the presence of ionizable carboxyl groups, pectin likely formed an external hydrated layer around the particles, conferring negative surface charge and contributing to electrostatic stabilization (Jonassen et al. 2013). LUT incorporation caused only a slight increase in surface negativity, indicating that the flavonoid did not represent the primary determinant of nanoparticle charge.

The encapsulation efficiency of LUT in ZPEC/LUT NP reached 87%, demonstrating the efficient incorporation of the hydrophobic flavonoid into the zein matrix. No significant loss of LUT was observed in the polymer‐free control after centrifugation, indicating that spontaneous sedimentation of LUT under the experimental conditions was negligible. This high encapsulation efficiency may be explained by the affinity between LUT and hydrophobic domains of zein, which favor compound retention during nanoparticle self‐assembly. During solvent displacement, the reduction of zein solubility promotes rapid formation of protein‐rich colloidal structures capable of entrapping hydrophobic molecules. Similar encapsulation efficiencies have been reported for zein‐based carriers containing poorly water‐soluble polyphenols and flavonoids, supporting the suitability of zein systems for nutraceutical delivery applications (Patel and Velikov 2014; Giteru et al. 2021; Li, Zhang, Chen, et al. 2023).

Overall, these findings indicate that the simplified zein–pectin formulation was capable of efficiently incorporating LUT while maintaining adequate physicochemical properties and colloidal stability.

3.2. Fourier Transform Infrared

FTIR spectroscopy was performed to investigate possible molecular interactions among LUT, zein, pectin, and the nanoparticle systems. The spectra of individual components and nanoparticle formulations are presented in Figure 3. LUT (Figure 3a) exhibited characteristic absorption bands commonly associated with flavonoid structures. A broad band around 3400 cm⁻¹ corresponded to O–H stretching vibrations from phenolic hydroxyl groups. Peaks near 1650 and 1600 cm⁻¹ were attributed to C = O stretching and aromatic C = C vibrations, respectively, whereas signals in the region of 1100–1300 cm⁻¹ were associated with C–O and ether‐related vibrations, consistent with LUT's polyphenolic structure (Elnaggar et al. 2018).

FIGURE 3.

FTIR Spectra of (a) LUT, (b) Pectin, (c) Zein, (d) ZPEC/LUT NP, and (e) ZPEC NP.

Pectin (Figure 3b) presented a broad O–H stretching band around 3400 cm⁻¹ and a characteristic peak near 1730–1750 cm⁻¹ associated with esterified carboxylic groups. The C–O–C stretching vibrations typical of glycosidic linkages appear around 1050–1150 cm⁻¹, consistent with the pectin backbone structure (Shinde et al. 2019). Zein (Figure 3c) exhibited typical protein bands, including amide I near 1650 cm⁻¹ and amide II around 1540 cm⁻¹, confirming the expected protein structure (Li et al. 2022).

The spectra of ZPEC NP (Figure 3e) and ZPEC/LUT NP (Figure 3d) exhibited highly similar spectral profiles. Both formulations retained the characteristic absorption regions associated with zein and pectin, indicating preservation of the biopolymeric matrix after nanoparticle formation. No substantial peak shifts or the appearance of new absorption bands was observed after LUT incorporation.

The high similarity between unloaded and loaded nanoparticle spectra may be explained by the relatively low proportion of LUT in the formulation and by overlap between LUT absorption bands and the broader signals associated with zein and pectin. This behavior suggests that LUT incorporation did not induce major structural changes in the nanoparticle matrix and likely occurred through physical entrapment and non‐covalent interactions rather than chemical modification. Similar findings have been reported in flavonoid‐loaded zein systems, where FTIR analysis showed limited spectral changes despite successful encapsulation (Palazzo et al. 2019; Li et al. 2022).

3.3. X‐Ray Diffraction

XRD analysis was performed to evaluate the crystalline or amorphous nature of LUT, the raw biopolymers, and the nanoparticle formulations (Figure 4). Pure LUT (Figure 4a) displays multiple sharp, intense peaks, especially in the 2θ range of 15°–30°, confirming its highly crystalline nature (Alshehri et al. 2020). In contrast, zein (Figure 4c) shows a broad peak between 2θ ≈ 10°–30°, typical of partially amorphous protein structures (Hajjari et al. 2021). Pectin (Figure 4b) also exhibits a diffuse halo, confirming its amorphous polysaccharide nature (Hoque et al. 2024). The physical mixture (Figure 4d) shows a superposition of features from the individual components, with crystalline peaks of LUT still visible. This result indicates that simple physical mixing was not sufficient to disrupt the crystalline structure of LUT.

FIGURE 4.

X‐ray diffraction patterns of (a) LUT, (b) pectin, (c) zein, (d) physical mixture, (e) ZPEC/LUT NP, and (f) ZPEC NP.

Both ZPEC NP (Figure 4f) and ZPEC/LUT NP (Figure 4e) exhibited broad amorphous diffraction patterns, with no clear crystalline peaks attributable to LUT. The similarity between ZPEC NP and ZPEC/LUT NP suggests that LUT incorporation did not substantially alter the overall amorphous structure of the zein–pectin matrix. Importantly, the disappearance of the characteristic crystalline peaks of LUT in ZPEC/LUT NP indicates that the flavonoid was no longer present as a crystalline phase after nanoparticle formation. This result suggests that LUT was molecularly dispersed or incorporated in an amorphous state within the zein–pectin matrix. Such amorphization is commonly observed after antisolvent precipitation and may contribute to improved apparent dispersibility of poorly water‐soluble bioactive compounds (Hajjari et al. 2021). Therefore, although XRD did not reveal major structural differences between unloaded and loaded nanoparticles, the absence of LUT crystalline peaks provides evidence of successful incorporation into the nanoparticle system.

3.4. Thermogravimetric Analysis

TGA was performed to investigate the thermal behavior of LUT, raw materials, physical mixture, and ZPEC/LUT NP (Figure 5). LUT (Figure 5a) showed an initial mass‐loss event near 59°C, followed by degradation events centered at approximately 284°C and 357°C. These thermal transitions are consistent with decomposition of the flavonoid structure and are characteristic of its crystalline organization (Diedrich et al. 2023). Pectin (Figure 5b) exhibited an initial thermal event around 70°C, associated with evaporation of adsorbed water, followed by a major degradation event centered at approximately 256°C. This behavior is commonly attributed to depolymerization and decomposition of polysaccharide chains (Shivangi et al. 2021). Zein (Figure 5c) displayed a first thermal event around 54°C related to moisture loss, followed by a major degradation peak at approximately 329°C. This profile is consistent with thermal decomposition of protein chains and agrees with previous reports for zein‐based materials (Magoshi et al. 1992).

FIGURE 5.

Thermogravimetric (TGA) and derivative thermogravimetric (DTG) curves of (a) LUT, (b) pectin, (c) zein, and (d) ZPEC/LUT NP.

The thermogram of ZPEC/LUT NP (Figure 5d) exhibited an initial event at approximately 50°C and a main degradation peak centered around 322°C. The nanoparticle formulation displayed a thermal profile more similar to zein than to free LUT, suggesting that the protein matrix dominated the thermal behavior of the system. Importantly, the characteristic thermal events observed for free LUT at 284°C and 357°C were no longer clearly distinguishable after nanoparticle formation. This result suggests that LUT was incorporated within the zein–pectin matrix and was no longer present as a separate crystalline phase. The absence of distinct LUT degradation events may indicate molecular dispersion or strong physical confinement within the nanoparticle structure.

These findings are consistent with XRD results, which showed disappearance of LUT crystalline peaks after nanoparticle formation, further supporting successful incorporation of LUT into the nanoparticle system.

3.5. Short‐Term Colloidal Stability Under Different Temperature Conditions

The short‐term colloidal stability of ZPEC/LUT NP was evaluated over 60 days under two storage conditions: room temperature and refrigeration at 10°C. Changes in mean particle diameter and PDI were monitored to investigate the influence of storage temperature on nanoparticle behavior (Figure 6).

FIGURE 6.

Short‐term colloidal stability of ZPEC/LUT NP during 60 days of storage. Mean particle diameter (a and b) and polydispersity index (c and d) were monitored at room temperature (∼25°C) and under refrigeration (10°C). Data are presented as mean ± SD (n = 3). Different lowercase letters indicate statistically significant differences among storage times within the same condition (p < 0.05).

The mean particle diameter remained within the nanometric range throughout the storage period under both conditions (Figure 6a,b). At room temperature, fluctuations in particle size were observed during storage, with values ranging approximately from 240 to 440 nm. In contrast, nanoparticles stored at 10°C exhibited comparatively smaller variations during the first 21 days, although a transient increase in particle size was observed at Day 28, followed by reduction at Day 60. Despite these fluctuations, no progressive increase in particle diameter was observed over time, suggesting the absence of pronounced aggregation phenomena. The reduction in particle size observed at later time points may reflect structural rearrangements within the colloidal system or changes in particle hydration behavior during storage.

The PDI results (Figure 6c,d) remained below approximately 0.35 under all evaluated conditions, indicating maintenance of acceptable colloidal homogeneity throughout the study period. Although some statistically significant differences were observed at specific time points, no marked increase in PDI was detected over storage, suggesting preservation of nanoparticle population distribution.

Overall, the findings indicate that ZPEC/LUT NP maintained short‐term colloidal stability during 60 days of storage under both room temperature and refrigerated conditions. The stabilizing effect may be associated with the hydrated pectin layer adsorbed onto the nanoparticle surface, which likely contributed to steric and electrostatic stabilization.

However, it should be emphasized that this study evaluated only short‐term colloidal stability under two temperature conditions. Long‐term storage behavior and the effects of additional environmental variables, including pH variation and light exposure, remain to be investigated in future studies.

3.6. In Vitro Simulated Digestion and Apparent Bioaccessibility of ZPEC/LUT NP

The gastrointestinal behavior of ZPEC/LUT NP was evaluated under simulated gastric and intestinal conditions to investigate the transfer of LUT into soluble fractions throughout digestion. Considering the sequential fractionation design employed in this study, the obtained values were interpreted as an estimate of apparent bioaccessibility rather than absolute physiological intestinal bioaccessibility.

As shown in Figure 7, only a limited amount of LUT was detected in the soluble fraction during the gastric stage, reaching approximately 8% after 2 h under SGF conditions (pH 1.2). This low transfer may be explained by the hydrophobic nature of LUT and the protective effect of the zein‐rich matrix. Under acidic conditions, zein tends to maintain a compact protein network that restricts rapid diffusion of hydrophobic compounds, thereby limiting LUT migration into the surrounding medium. In addition, LUT exhibits poor aqueous solubility under gastric conditions, which may further reduce its transfer into the soluble phase.

FIGURE 7.

Apparent bioaccessibility of LUT from ZPEC/LUT NP during in vitro gastrointestinal digestion. SGF (pH 1.2, 0–2 h) followed by SIF (pH 6.8, 2–6 h). Values represent the soluble fraction (mean ± SD, n = 3). SGF, simulated gastric fluid; SIF, simulated intestinal fluid.

After gastric digestion, the precipitated fraction was subjected to intestinal conditions in the presence of pancreatin and bile salts. During this stage, a gradual increase in LUT recovery in the soluble fraction was observed, reaching approximately 16% after completion of digestion. Digestive enzymes and bile salts may contribute to matrix disassembly and facilitate the incorporation of hydrophobic compounds into soluble fractions present in the digestion medium, favoring their transfer into the aqueous phase.

Interestingly, the increase observed during the intestinal stage was progressive, and no burst release profile was detected. This behavior suggests that LUT remained strongly associated with the zein–pectin structure throughout digestion. Hydrophobic interactions between LUT and zein, together with intermolecular interactions within the nanoparticle matrix, may have delayed LUT diffusion and promoted sustained release behavior.

Although the apparent bioaccessibility observed for ZPEC/LUT NP was moderate, the results indicate that the nanoparticles were able to preserve LUT during acidic conditions and modulate its transfer under intestinal environments. Similar controlled gastrointestinal behavior has been reported for zein‐based delivery systems containing hydrophobic compounds, where strong matrix interactions promoted delayed release and gradual transfer into soluble phases (Penalva et al. 2017; Li, Zhang, Chen, et al. 2023).

It should also be considered that the fractionation procedure adopted in this study involved separation of soluble and precipitated fractions after the gastric stage. Consequently, LUT present in the gastric soluble fraction did not proceed to intestinal digestion. Therefore, the obtained values should be interpreted within the context of this experimental design and may underestimate the total amount of LUT potentially available after complete gastrointestinal transit.

Overall, the results demonstrate that ZPEC/LUT NP promoted a gradual transfer of LUT into soluble fractions and exhibited controlled gastrointestinal behavior, suggesting that the zein–pectin matrix may provide protection and sustained release characteristics for hydrophobic flavonoids.

3.7. Evaluation of Antioxidant Activity

The antioxidant activity of free LUT, ZPEC NP, and ZPEC/LUT NP was evaluated using the ABTS radical scavenging assay, and the results are presented in Figure 8. For comparative purposes, the amount of free LUT and LUT incorporated in ZPEC/LUT NP was normalized to equivalent LUT concentrations (6.3–50 µg/mL). In the case of ZPEC NP, equivalent nanoparticle concentrations were used.

FIGURE 8.

ABTS radical scavenging activity of LUT, ZPEC NP, and ZPEC/LUT NP. Equivalent LUT concentrations (6.3–50 µg/mL) were used for comparison. For ZPEC NP, equivalent nanoparticle concentrations were employed. Different letters indicate statistically significant differences among formulations at the same concentration (p < 0.05). ZPEC NP, zein–pectin nanoparticle.

All formulations exhibited concentration‐dependent antioxidant activity, with increasing inhibition values observed at higher concentrations. Free LUT showed progressive radical scavenging activity, reaching approximately 77% inhibition at 50 µg/mL. At concentrations of 12.5, 25.0, and 50.0 µg/mL, ZPEC/LUT NP showed significantly higher ABTS radical scavenging activity than free LUT (p < 0.05), reaching approximately 93% at the highest concentration. At 6.3 µg/mL, although ZPEC/LUT NP showed a higher mean inhibition value, the overlap of SDs suggests that this difference should be interpreted with caution.

ZPEC NP exhibited comparatively low inhibition values, ranging from approximately 8%–25%. Although markedly lower than ZPEC/LUT NP, these findings suggest that the carrier matrix itself contributed to the overall antioxidant response. Previous studies have demonstrated that zein‐based nanoparticle systems may display intrinsic antioxidant behavior due to amino acid residues capable of interacting with radical species. Similar trends have been reported in zein systems loaded with polyphenols, such as EGCG (Jin et al. 2022), ellagic acid (de Souza Tavares et al. 2023), and anacardic acid (Araújo et al. 2020), in which both the carrier and encapsulated compounds contributed to radical scavenging behavior.

Therefore, the enhanced antioxidant activity observed for ZPEC/LUT NP should not be interpreted as a synergistic effect between LUT and nanoparticle components. Considering that ZPEC NP already exhibited measurable ABTS inhibition, the observed response likely reflects additive contributions from LUT and the carrier system. In addition, nanoencapsulation may improve apparent antioxidant performance by increasing LUT dispersion and facilitating contact with radicals in aqueous media rather than increasing the intrinsic antioxidant capacity of LUT molecules.

It is important to note that the ABTS assay has limitations when applied to protein‐based systems because proteins may directly interact with radical species. Thus, although the results indicate preservation and enhancement of antioxidant performance after nanoencapsulation, interpretation should consider the potential contribution of zein to the observed effect.

The IC₅₀ values further supported the improved apparent antioxidant performance of the nanoencapsulated system. ZPEC/LUT NP exhibited an IC₅₀ of approximately 12.5 µg/mL, which was lower than that of free LUT (22.5 µg/mL). This result suggests that nanoencapsulation enhanced the effective radical scavenging response of LUT under the aqueous conditions of the ABTS assay, likely by improving its dispersion and accessibility to ABTS radicals. In contrast, the IC₅₀ for ZPEC NP was not reached within the tested concentration range, indicating that although the carrier matrix contributed to antioxidant activity, its effect was considerably weaker than that of LUT‐containing formulations. Therefore, the improved activity of ZPEC/LUT NP should be interpreted as an enhancement of apparent antioxidant performance rather than evidence of a synergistic effect.

Overall, these findings indicate that the ZPEC NP system preserved LUT antioxidant properties while improving its apparent radical scavenging performance.

4. Conclusion

In this study, ZPEC/LUT NPs were successfully developed as a food‐grade platform for LUT encapsulation using the antisolvent precipitation method. The resulting nanoparticles exhibited suitable physicochemical properties, high encapsulation efficiency, and satisfactory colloidal stability during storage. Comparative evaluation of unloaded and LUT‐loaded nanoparticles demonstrated that LUT incorporation did not substantially alter particle morphology, size distribution, or surface charge, supporting formation of a stable zein–pectin system. FTIR, TG/DTG, and XRD analyses collectively suggested that LUT was physically incorporated into the nanoparticle matrix and predominantly present in an amorphous or molecularly dispersed state. These findings indicate successful encapsulation without evidence of major structural disruption or chemical modification of the biopolymeric carrier. Simulated gastrointestinal digestion demonstrated gradual transfer of LUT into soluble fractions throughout digestion, reaching an apparent bioaccessibility of approximately 16% after 6 h. Although the observed values were moderate, the nanoparticles promoted controlled LUT release and preserved the flavonoid during gastric conditions, suggesting a protective role of the zein–pectin matrix. Nanoencapsulation improved the apparent antioxidant performance of LUT, with ZPEC/LUT NP showing enhanced ABTS radical scavenging activity and lower IC₅₀ values compared with free LUT. This effect was primarily associated with improved aqueous dispersion and accessibility of LUT after encapsulation, whereas the zein–pectin matrix also contributed to the overall antioxidant response. Importantly, the improved activity should be interpreted as an enhancement of functional performance rather than evidence of synergistic interactions. Overall, these findings demonstrate that pectin‐stabilized zein nanoparticles represent a promising food‐grade strategy for improving the physicochemical and functional behavior of hydrophobic flavonoids. Future studies involving long‐term stability, cellular models, and in vivo investigations will be important to better understand the biological relevance and translational potential of this delivery system for food and nutraceutical applications.

Author Contributions

Natália Valendolf Pires: methodology, investigation, formal analysis, writing – original draft. Ariane Krause Padilha Lorenzett: visualization, writing – review and editing. Rossana Gabriela Del Jesus Vásquez Marcano: methodology, investigation. Jeferson Ziebarth: investigation, methodology. Rubiana Mara Mainardes: conceptualization, funding acquisition, writing – review and editing, project administration.

Funding

The authors thank to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES—Brazil) (Code 001) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq‐Brazil—proc 313800/2018‐9) for partial financial support.

Conflicts of Interest

The authors declare no conflicts of interest.