Preparation of Type-A Gelatin/Poly-γ-Glutamic Acid Nanoparticles for Enhancing the Stability and Bioavailability of (-)-Epigallocatechin Gallate

(-)-Epigallocatechin gallate (EGCG) has gained considerable attention owing to its beneficial properties. However, its application as a functional food is restricted due to its instability and low bioavailability. In the present study, a food-derived nanoparticle system based on type A gelatin/γ-PGA was developed to preserve and deliver EGCG. The EGCG/gelatin/γ-PGA nanoparticles had a particle size of 155.1 ± 7.3 nm with a zeta potential of −23.9 ± 0.9 mV. Moreover, the EGCG/gelatin/γ-PGA nanoparticles enhanced the long-term storage stability and sustained antioxidant activity of EGCG compared to EGCG/gelatin nanoparticles. The nanoparticles protected EGCG in simulated gastric fluid containing pepsin while releasing it in simulated intestinal fluid. Additionally, the amount of EGCG transported in the Caco-2 monolayers treated with EGCG/gelatin/γ-PGA nanoparticles was three times higher than that of free EGCG, which might be related to the paracellular pathway and endocytosis. These results suggest that EGCG/gelatin/γ-PGA nanoparticles might be an effective delivery vehicle for EGCG, enhancing its potential applications in the functional food field.


Introduction
(-)-Epigallocatechin gallate (EGCG) is the most prevalent catechin component in green tea [1]. EGCG has strong oxidation resistance due to the substantial number of phenolic hydroxyl groups in its structure [2]. It has been demonstrated that EGCG regulates metabolism in the human body [3] and has anti-tumor [4] and anti-cardiovascular disease properties [5]. Because of these health advantages, it has drawn special interest in recent years and is considered a potential alternative to synthetic food additives [6] or commercial high-dose food supplements [7]. However, practical applications of EGCG are limited by several factors. First, EGCG is less resilient to temperature, light, pH, and oxygen, which significantly accelerates its breakdown during long-term storage [8]. Additionally, EGCG may be destroyed by digestive enzymes and other biologically active substances in gastrointestinal fluids after ingestion due to its hostile environment in humans [9]. Furthermore, the intestine prolongs the residence time of EGCG, resulting in insufficient EGCG permeability [10]. These defects significantly reduce the stability and bioavailability of EGCG, severely limiting its potential applications [11]. Therefore, an efficient EGCG delivery system is required.
Small intestinal epithelial cells can be penetrated by nanoparticles through either paracellular or endocytic pathways [12,13]. Therefore, the development of EGCG-loaded nanocarriers is crucial for overcoming the instability and low bioavailability of EGCG. EGCG has been encapsulated in multiple nanodelivery systems [14,15], including nanoparticles [16] and nanoemulsions [17]. Nanoparticles with proteins and polysaccharides as wall materials have been widely used in the production of nanocarrier systems due to their

Characterization of the Produced Nanoparticles
The particle size distribution, polydispersity index (PDI), and zeta potential of the nanoparticles were measured using a Zetasizer Nano (Malvern ZS90, UK) and dynamic light scattering (DLS) techniques. Fourier-transform infrared (FT-IR) spectroscopy was used to analyze the chemical structures of EGCG, type-A gelatin, γ-PGA, and the nanoparticles within the range of 500 to 4000 cm −1 .
The fluorescence spectra of the nanoparticles were recorded using a fluorescence spectrophotometer at two different temperatures (25 • C and 37 • C). The nanoparticle solution was diluted ten times with phosphate buffer (pH 6). The excitation wavelength was set to 280 nm, and the emission range was 300-500 nm, with both excitation and emission slit widths set to 5 nm.
The apparent morphology of the two types of nanoparticles was confirmed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM samples were freeze-dried for 48 h and sputter-coated with gold for 120 s before observation (Hitachi S4800, Tokyo, Japan). The TEM samples were prepared by depositing a drop of the nanoparticle suspension onto a carbon-coated copper grid with a mesh size of 400, followed by removing surface water using filter paper and drying with air. The dried samples were then examined using transmission electron microscopy (TEM) (Hitachi H-600, Japan).

Assessments of Encapsulation Efficiency of EGCG in Nanoparticles
The prepared small-particle-size nanoparticles were collected via high-speed centrifugation (15,000 rpm, 4 • C, 15 min). The supernatant was then filtered through a 0.22 µm membrane, and the amount of free EGCG was assessed via high-performance liquid chromatography (HPLC) [34]. Briefly, an Agilent 1260 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a UV detector (SPD-20A) and a Sepax GP-C18 column (4.6 mm × 250 mm) was utilized. The mobile phase consisted of ddH 2 O, ACN, and TFA (919/80/1, v/v) for Phase A and ddH 2 O, ACN, MeOH, and TFA (699/270/30/1, v/v) for Phase B with a system flow rate of 1.2 mL/min. The injection volume was 10 µL, and the column oven temperature was set to 25 • C. Gradient elution was performed as follows: 95/5 at 0 min, 30/70 at 1.5 min (convex), 1/99 at 3 min (convex), and 95/5 at 3-5 min (step immediate). The total chromatographic runtime was 9.5 min. Individual EGCG content was quantified by constructing multilevel calibration curves (EGCG concentrations: 25, 50, 75, 100, 150, and 200 ppm) from the response at 280 nm caused by the injection of authentic EGCG standards. Total EGCG was computed as the sum of all individual EGCG measurements. The EGCG encapsulation efficiency (EE) and loading capacity (LC) of the nanoparticles were calculated using the following formulas: Loading capacity (%) = ( where E 1 and E 2 are the total amounts of EGCG and free EGCG as determined by HPLC, respectively, and E 3 is the weight of the nanoparticles.

Storage Stability of EGCG/Gelatin/γ-PGA Nanoparticles
The nanoparticle suspensions were stored in a dark place at 4 • C and 25 • C for 28 d. The particle size and PDI of the nanoparticles were measured every 7 d. Before testing for nanoparticle dispersion, each sample was ultrasonicated for 1 min at 20 kHz and 400 W. The experiments were performed in triplicate.

EGCG Release of Nanoparticles under Different pH Conditions
To investigate the pH sensitivity of the nanoparticles, they were dispersed in buffer solutions with varying pH values. First, the EGCG/gelatin/γ-PGA nanoparticle suspensions were treated with high-speed centrifugation (15,000 rpm, 4 • C, 15 min), and the sediments were then ultrasonically dispersed in buffer solutions. The pH was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, and 7.5 using 0.1 M HCl or NaOH solution. The EGCG/gelatin/γ-PGA nanoparticle suspensions were then incubated at 37 • C in a water bath for 2 h. After incubation, 2.0 mL of the sample suspensions were collected and centrifuged to obtain the supernatant. The amount of EGCG released was evaluated using HPLC by measuring the EGCG content in the supernatant. The percentage decline in the EGCG content of the nanoparticles was used to calculate the amount of EGCG released after centrifugation. All experiments were performed in triplicate.

EGCG Release
where E 1 and E 2 represent the total amounts of EGCG and free EGCG, respectively.

In Vitro Release of the Complex Nanoparticles
In vitro simulated digestion experiments were conducted using simulated gastric fluid (SGF, pH 2.1, with pepsin) and simulated intestinal fluid (SIF, pH 7.2, with trypsin). The nanoparticles were incubated in SGF and SIF for 0-4 h, and the amount of EGCG released was measured using HPLC. The centrifuged nanoparticles were dispersed in SGF or SIF (0 h) and magnetically stirred (200 rpm) in a 37 • C water bath. The amount of EGCG in the supernatant was quantified at various time points (1, 2, 3, and 4 h) using HPLC.

Antioxidant Measurements of Complex Nanoparticles by DPPH/ABTs
The free radical scavenging activity of the EGCG/gelatin and EGCG/gelatin/γ-PGA nanoparticles against DPPH was measured using the DPPH scavenging assay kit [35]. Briefly, 0.4 mL of the sample solution was mixed with 0.6 mL of DPPH working solution and left in the dark at 25 • C for 30 min before centrifugation at 4000 rpm for 5 min. The absorbance of the reaction solution was measured at 517 nm using an enzyme-labeled instrument. The DPPH radical scavenging activities of the different samples were calculated using the following formula: Here, A c represents the absorbance of the control (sample solution mixed with 80% methanol), A s represents the absorbance of the sample (sample solution mixed with working solution), and A b represents the absorbance of the blank (80% methanol mixed with working solution).
To assess ABTS radical scavenging activity, an ABTS total antioxidant capacity test kit was used [36]. Firstly, the ABTS solution in the kit was mixed with an equal volume of oxidant solution and allowed to react for 16 h at 25 • C in the dark to prepare the ABTS working solution. Next, the ABTS working solution (1 mL) was mixed with 80% ethanol (99 mL) to obtain a fresh ABTS solution with an absorbance of approximately 0.7 at 734 nm. Then, the sample solution (10 µL) and fresh ABTS solution (200 µL) were added sequentially and reacted at 25 • C for 6 min. The absorbance of the samples was immediately measured at 734 nm. The ABTS radical scavenging activity of the samples was calculated using the following formula: where A s represents the absorbance of the sample at 734 nm and A b represents the blank (distilled water) absorbance at 734 nm.

EGCG Transportation on Caco-2 Cell Monolayers
Caco-2 cells were cultured on 12-well transwell plates (Corning Inc., Corning, NY, USA) with 0.4 µm aperture, 12 mm inner diameter, and 1.12 cm 2 growth area. The seeding density was 3 × 10 5 cells, and the cells were maintained in Caco-2 cell-specific medium (Yifeixue) in the donor and receiver compartments to promote cell growth. The medium was changed every 48 h for the first week and every 24 h thereafter. The cells were incubated at 37 • C with 95% air and 5% CO 2 for 18-21 days, and the transepithelial electrical resistance (TEER) was measured to be over 400 Ω cm 2 before conducting the experiments.
The transportation study was conducted with minor adjustments to a previously published protocol [37]. TEER values of the Caco-2 monolayers were measured using a Millipore Millicell ERS-2 Cell Resistor (Millipore Corp., Burlington, MA, USA). The transport medium for permeation experiments was HBSS (pH 6.2). Before the experiment, Caco-2 monolayers were rinsed twice and adaptively cultured for 30 min in the transport medium. Free EGCG, EGCG/gelatin nanoparticles, and EGCG/gelatin/γ-PGA nanoparticles were added as the donor medium to maintain the EGCG concentration at 0.025 mg/mL. Caco-2 monolayers were then treated with the test solutions (0.5 mL) in the donor compartment and 1.5 mL of the transport medium (HBSS, pH 6.2) in the receiver compartment. The control group was treated with equal volumes of HBSS in the donor compartment. The plates were incubated on a shaker at 150 rpm at 37 • C. Samples (50 µL) were collected from the receiving compartment at different time intervals (30, 60, 90, and 120 min), and HPLC analysis was used to measure the EGCG concentrations [34]. The cumulative transport rate of EGCG was calculated and plotted against time.
The integrity of the Caco-2 monolayers was maintained by measuring the TEER value before each experiment, which was calculated as follows: where TEER (Ω) is the transmembrane resistance of Caco-2 monolayers, read directly from the voltmeter, TEER b (Ω) is the background that transwell plates (without cells) supplemented with Caco-2 cell-specific medium. The area (cm 2 ) was the membrane area of the transwell plates, 1.12 cm 2 .

Statistical Analysis
All experiments were conducted in triplicate. Single-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) version 18.0 (Chenna Inc., Chicago, IL, USA) was performed to examine the variations in samples, with a significance level of α = 0.05. Post hoc tests were conducted where appropriate. Data are presented as means ± standard deviations (SDs).

Preparation of Complex Nanoparticles
The use of gelatin and γ-PGA for the preparation of EGCG/gelatin/γ-PGA nanoparticles was based on their food safety and biocompatibility. The formation of nano complexes was achieved through the electrostatic interaction between γ-PGA and gelatin, while stable EGCG nanoparticles were formed via hydrogen bonding with gelatin [33]. To ensure the stability and optimal biological properties of the nanoparticles, it is important to investigate the impact of their physicochemical characteristics, particularly the mass ratio of their components, on the final product. Therefore, in this study, we evaluated the effect of the mass ratio of the components on the physical properties of EGCG/gelatin/γ-PGA nanoparticles.
Initially, we investigated the characteristics of nanoparticles that were prepared using different gelatin/γ-PGA mass ratios, ranging from 1:1 to 8:1, as presented in Table 1. The levels of EGCG and γ-PGA were maintained at 1 mg/mL throughout the study. With increasing concentrations of gelatin, the size and polydispersity index (PDI) of gelatin/γ-PGA nanoparticles gradually increased. Above a gelatin concentration of 6 mg/mL, we observed aggregation and precipitation of nanoparticles. This outcome is likely due to gelatin neutralizing the negative surface charge of the nanoparticles, thereby reducing their physical stability. Furthermore, the average particle size of EGCG/gelatin/γ-PGA nanoparticles was larger, and their absolute zeta potential was lower than that of gelatin/γ-PGA nanoparticles, indicating that EGCG was not simply embedded within the nanoparticles. All the nanoparticles had negative zeta potentials, and this is likely due to the phenol hydroxyl group of EGCG being slightly deprotonated at pH 6.2 [38] and the side-chain carboxylate group of γ-PGA being more ionized in weak acid solution because its pKa value is near 2.5 [39]. This is the primary reason for the negative charges of EGCG/gelatin/γ-PGA nanoparticles. Zeta potential has long been considered a reliable indicator for evaluating the stability of colloids [40]. In general, nanoparticles with zeta potentials above +30 mV or below −30 mV are deemed stable [41]. The EGCG/gelatin/γ-PGA nanoparticles had a higher absolute zeta potential than the EGCG/gelatin nanoparticles, indicating their greater stability in suspension [42]. Table 1. Mean particle sizes, PDI, and zeta potential of EGCG/gelatin/γ-PGA and gelatin/γ-PGA nanoparticles (n = 3 batches) a (p < 0.05).  Based on the previous experiments, the mass ratio of gelatin/γ-PGA at 4:1 was found to produce nanoparticles with favorable properties. Hence, the concentrations of gelatin and γ-PGA were fixed at 4 mg/mL and 1 mg/mL, respectively, and mixed with EGCG solutions of different concentrations. The mixture became increasingly turbid as the EGCG concentration increased ( Figure 1A). The particle size and zeta potential of nanoparticles produced with varying EGCG/gelatin mass ratios are shown in Figure 1B. It was observed that EGCG concentration had little effect on the nanoparticles, and they maintained particle sizes of approximately 150 nm and an absolute zeta potential of 23 mV in most cases. However, when the EGCG/gelatin mass ratio was 1:1, the size of EGCG/gelatin/γ-PGA nanoparticles increased to 233 nm. Similarly, EGCG/gelatin/γ-PGA nanoparticles had larger particle sizes and absolute zeta potentials compared to EGCG/gelatin nanoparticles. While the majority of EGCG was located inside the nanoparticles, some may have bound to gelatin at the surface, affecting the potential of the nanoparticles. The EGCG/gelatin nanoparticles had a negative charge of zeta potentials at pH 6.2, and the absolute zeta potential decreased with increasing EGCG concentration, as the pKa value of EGCG was approximately 7.68. The encapsulation efficiency (EE) and loading capacity (LC) of EGCG/gelatin/γ-PGA nanoparticles were between 51.2-72.4% and 0.75-1.06%, respectively ( Figure 1C). The highest EE was observed when the EGCG concentration was 2 mg/mL. However, as the concentration of EGCG mixed with the γ-PGA aqueous solution increased, the EE and LC of EGCG decreased. This was because both the EGCG content in the original mixture and the EGCG concentration outside the nanoparticles had increased. The encapsulation efficiency of EGCG is influenced by the partition between the nanoparticle space and the surrounding aqueous environment [43]. The high hydrophilicity of γ-PGA may explain the increase in encapsulation efficiency of EGCG/gelatin/γ-PGA nanoparticles. and γ-PGA were fixed at 4 mg/mL and 1 mg/mL, respectively, and mixed with EGCG solutions of different concentrations. The mixture became increasingly turbid as the EGCG concentration increased ( Figure 1A). The particle size and zeta potential of nanoparticles produced with varying EGCG/gelatin mass ratios are shown in Figure 1B. It was observed that EGCG concentration had little effect on the nanoparticles, and they maintained particle sizes of approximately 150 nm and an absolute zeta potential of 23 mV in most cases. However, when the EGCG/gelatin mass ratio was 1:1, the size of EGCG/gelatin/γ-PGA nanoparticles increased to 233 nm. Similarly, EGCG/gelatin/γ-PGA nanoparticles had larger particle sizes and absolute zeta potentials compared to EGCG/gelatin nanoparticles. While the majority of EGCG was located inside the nanoparticles, some may have bound to gelatin at the surface, affecting the potential of the nanoparticles. The EGCG/gelatin nanoparticles had a negative charge of zeta potentials at pH 6.2, and the absolute zeta potential decreased with increasing EGCG concentration, as the pKa value of EGCG was approximately 7.68. The encapsulation efficiency (EE) and loading capacity (LC) of EGCG/gelatin/γ-PGA nanoparticles were between 51.2-72.4% and 0.75-1.06%, respectively ( Figure 1C). The highest EE was observed when the EGCG concentration was 2 mg/mL. However, as the concentration of EGCG mixed with the γ-PGA aqueous solution increased, the EE and LC of EGCG decreased. This was because both the EGCG content in the original mixture and the EGCG concentration outside the nanoparticles had increased. The encapsulation efficiency of EGCG is influenced by the partition between the nanoparticle space and the surrounding aqueous environment [43]. The high hydrophilicity of γ-PGA may explain the increase in encapsulation efficiency of EGCG/gelatin/γ-PGA nanoparticles.

Characterization of Complex Nanoparticles
The EGCG/gelatin/γ-PGA nanoparticles were produced by mixing a solution of γ-PGA (1 mg/mL) and EGCG (2 mg/mL) with a solution of type-A gelatin (4 mg/mL) at room temperature (25 • C) while stirring. The FT-IR absorption spectrums of EGCG, gelatin, γ-PGA, EGCG/gelatin nanoparticles, and EGCG/gelatin/γ-PGA nanoparticles were shown in Figure 2A. The absorption band at approximately 3352 cm −1 in EGCG was due to the vibrating O-H bond of the phenolic hydroxyl group. Gelatin showed strong amide absorptions, with the amide I C=O stretching band at 1642 cm −1 and the amide II N-H bending band at 1534 cm −1 [44]. γ-PGA exhibited strong absorption peaks at 1634 cm −1 due to the carboxyl group ion (-COO − ) [12]. In the spectra of the gelatin/EGCG nanoparticles, the O-H peak of the phenolic hydroxyl group at 3352 cm −1 was red-shifted to 3306 cm −1 and became broader than that of EGCG. Other shifts in the characteristic peaks of EGCG/gelatin/γ-PGA nanoparticles, such as 3285 cm −1 , 1541 cm −1 , and 1640 cm −1 , were also found, attributed to the phenolic hydroxyl of EGCG, the amide II of gelatin, and the carboxyl group of γ-PGA. These results suggested that the formation of nanoparticles was induced by electrostatic interactions and hydrogen bonding.
in Figure 2A. The absorption band at approximately 3352 cm −1 in EGCG was due to the vibrating O-H bond of the phenolic hydroxyl group. Gelatin showed strong amide absorptions, with the amide I C=O stretching band at 1642 cm −1 and the amide II N-H bending band at 1534 cm −1 [44]. γ-PGA exhibited strong absorption peaks at 1634 cm −1 due to the carboxyl group ion (-COO − ) [12]. In the spectra of the gelatin/EGCG nanoparticles, the O-H peak of the phenolic hydroxyl group at 3352 cm −1 was red-shifted to 3306 cm −1 and became broader than that of EGCG. Other shifts in the characteristic peaks of EGCG/gelatin/γ-PGA nanoparticles, such as 3285 cm −1 , 1541 cm −1 , and 1640 cm −1 , were also found, attributed to the phenolic hydroxyl of EGCG, the amide II of gelatin, and the carboxyl group of γ-PGA. These results suggested that the formation of nanoparticles was induced by electrostatic interactions and hydrogen bonding.  The morphology of the nanoparticles was analyzed using SEM and TEM techniques ( Figure 2B). SEM images demonstrated that both types of nanoparticles were spherical in shape, with some adhesion between them, possibly due to the interaction between gelatin, EGCG, and γ-PGA. TEM results showed that the EGCG/gelatin/γ-PGA nanoparticles had a larger particle size than EGCG/gelatin nanoparticles, which was consistent with the particle size measurements obtained earlier. These findings confirmed the successful encapsulation of EGCG in the gelatin/γ-PGA nanoparticles.
Fluorescence spectroscopy was used to investigate the interactions between gelatin, EGCG, and γ-PGA at different temperatures ( Figure 2C,D). Gelatin fluorescence is mainly attributed to the intrinsic Tyr fluorophore, while Phe cannot be excited in most cases [45]. Additionally, γ-PGA containing only glutamic acid does not exhibit fluorescence. The EGCG/gelatin/γ-PGA nanoparticles were red-shifted as the gelatin/γ-PGA mass ratio changed from 1:8 to 1:1 at 25 • C, indicating that EGCG bound to the protein and changed its structure. The maximum emission wavelength of the EGCG/gelatin nanoparticles remained constant, indicating that there were no significant changes in the protein conformation. The fluorescence intensity of EGCG/gelatin/γ-PGA nanoparticles was higher than that of EGCG/gelatin nanoparticles at both 25 • C and 37 • C, and the fluorescence intensity of nanoparticles decreased with increasing EGCG concentration, which could be attributed to the fluorescence quenching of the interaction between Trp and EGCG. These findings suggest that EGCG interacted with gelatin in the nanoparticles, resulting in a reduction in fluorescence intensity.

Storage Stability of EGCG/gelatin/γ-PGA Nanoparticles
Despite the promising therapeutic effects of EGCG, its poor stability has hindered its large-scale production and use in the food industry. The effect of temperature on the stability of nanoparticles during storage was investigated (Figure 3). The particle size of EGCG/gelatin/γ-PGA nanoparticles remained constant throughout the storage period, with an average particle size of approximately 160 nm after 28 days. The PDI was maintained between 0.2 and 0.6, indicating that the EGCG/gelatin/γ-PGA nanoparticles were stable and did not significantly aggregate. The particle size and PDI of the EGCG/gelatin nanoparticles slowly increased during storage, which could be attributed to nanoparticle aggregation and swelling in the solution. Additionally, all nanoparticles exhibited lower PDI at 4 • C, and the average particle size of EGCG/gelatin nanoparticles was 196.9 nm, which was smaller than the 250.9 nm at 25 • C. These findings suggest that the EGCG/gelatin/γ-PGA nanoparticles have excellent stability for long-term storage at 4 • C.
EGCG/gelatin/γ-PGA nanoparticles were red-shifted as the gelatin/γ-PGA mass rat changed from 1:8 to 1:1 at 25 °C, indicating that EGCG bound to the protein and change its structure. The maximum emission wavelength of the EGCG/gelatin nanoparticles r mained constant, indicating that there were no significant changes in the protein confo mation. The fluorescence intensity of EGCG/gelatin/γ-PGA nanoparticles was higher tha that of EGCG/gelatin nanoparticles at both 25 °C and 37 °C, and the fluorescence intensi of nanoparticles decreased with increasing EGCG concentration, which could be a tributed to the fluorescence quenching of the interaction between Trp and EGCG. The findings suggest that EGCG interacted with gelatin in the nanoparticles, resulting in reduction in fluorescence intensity.

Storage Stability of EGCG/gelatin/γ-PGA Nanoparticles
Despite the promising therapeutic effects of EGCG, its poor stability has hindered i large-scale production and use in the food industry. The effect of temperature on the st bility of nanoparticles during storage was investigated (Figure 3). The particle size EGCG/gelatin/γ-PGA nanoparticles remained constant throughout the storage perio with an average particle size of approximately 160 nm after 28 days. The PDI was mai tained between 0.2 and 0.6, indicating that the EGCG/gelatin/γ-PGA nanoparticles we stable and did not significantly aggregate. The particle size and PDI of the EGCG/gelat nanoparticles slowly increased during storage, which could be attributed to nanopartic aggregation and swelling in the solution. Additionally, all nanoparticles exhibited low PDI at 4 °C, and the average particle size of EGCG/gelatin nanoparticles was 196.9 nm which was smaller than the 250.9 nm at 25 °C. These findings suggest that the EGCG/ge atin/γ-PGA nanoparticles have excellent stability for long-term storage at 4 °C.

In Vitro Bioaccessibility of EGCG in EGCG/Gelatin/γ-PGA Nanoparticles
The harsh environment of the gastrointestinal tract, including elevated pH and metabolic enzymes, is one of the factors that hamper EGCG oral bioavailability [2]. The stability of the produced EGCG/gelatin/γ-PGA nanoparticles was assessed over a range of pH values by resuspending them in buffer solutions. Figure 4A shows the release rates of EGCG from the nanoparticles after 2 h of incubation in different pH solutions. The release rate of EGCG gradually increased with increasing pH, being only 14.2% in the pH 3-4 solution but reaching 74.9% in the weak alkaline solution (pH 7.5). This may be attributed to the reduced positive charge exhibited by type-A gelatin in a weakly alkaline solution, leading to the disintegration of nanoparticles and massive release of EGCG. Furthermore, the EGCG release rate of EGCG/gelatin/γ-PGA nanoparticles was lower than that of EGCG/gelatin nanoparticles in acidic or neutral solutions but higher in weakly alkaline solutions. This may be due to the increased stability of the EGCG/gelatin/γ-PGA complex. These results indicate that EGCG/gelatin/γ-PGA nanoparticles are more stable in an acidic environment, offering better protection for EGCG.
To investigate the effect of proteases on the nanoparticles during digestion, the prepared EGCG/gelatin/γ-PGA nanoparticles were exposed to simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) for four hours, and high-performance liquid chromatography (HPLC) was utilized to measure the release of EGCG at different time intervals (0.5, 1, 2, 3, and 4 h). As depicted in Figure 4B, EGCG/gelatin/γ-PGA nanoparticles remained stable in SGF, with a maximum EGCG release rate of 21.4% after four hours of incubation. Within the first 30 min, EGCG was rapidly released from the nanoparticles into the SIF at a rate that eventually reached 40%. The cumulative release rate of EGCG decreased slightly from 0.5-3 h, totaling 81%. From 3-4 h, the release rate of EGCG decreased sharply, and the cumulative release peaked at 84%. These results indicate that when exposed to pepsin in SGF, the nanoparticles remained relatively stable. However, in alkaline SIF, a large number of nanoparticles disintegrated, and EGCG was sustainably released. This can be attributed to the electrostatic interaction between gelatin and PGA, which hinders the contact between gelatin and protease. The high cumulative release rate suggests that EGCG/gelatin/γ-PGA nanoparticles serve as superior EGCG delivery carriers.

In Vitro Bioaccessibility of EGCG in EGCG/Gelatin/γ-PGA Nanoparticles
The harsh environment of the gastrointestinal tract, including elevated pH and me abolic enzymes, is one of the factors that hamper EGCG oral bioavailability [2]. The sta bility of the produced EGCG/gelatin/γ-PGA nanoparticles was assessed over a range o pH values by resuspending them in buffer solutions. Figure 4A shows the release rates o EGCG from the nanoparticles after 2 h of incubation in different pH solutions. The releas rate of EGCG gradually increased with increasing pH, being only 14.2% in the pH 3solution but reaching 74.9% in the weak alkaline solution (pH 7.5). This may be attribute to the reduced positive charge exhibited by type-A gelatin in a weakly alkaline solution leading to the disintegration of nanoparticles and massive release of EGCG. Furthermor the EGCG release rate of EGCG/gelatin/γ-PGA nanoparticles was lower than that o EGCG/gelatin nanoparticles in acidic or neutral solutions but higher in weakly alkalin solutions. This may be due to the increased stability of the EGCG/gelatin/γ-PGA comple These results indicate that EGCG/gelatin/γ-PGA nanoparticles are more stable in an acid environment, offering better protection for EGCG. To investigate the effect of proteases on the nanoparticles during digestion, the pr pared EGCG/gelatin/γ-PGA nanoparticles were exposed to simulated gastric fluid (SGF and simulated intestinal fluid (SIF) for four hours, and high-performance liquid chroma tography (HPLC) was utilized to measure the release of EGCG at different time interva (0.5, 1, 2, 3, and 4 h). As depicted in Figure 4B, EGCG/gelatin/γ-PGA nanoparticles r mained stable in SGF, with a maximum EGCG release rate of 21.4% after four hours o incubation. Within the first 30 min, EGCG was rapidly released from the nanoparticle into the SIF at a rate that eventually reached 40%. The cumulative release rate of EGC decreased slightly from 0.5-3 h, totaling 81%. From 3-4 h, the release rate of EGCG d creased sharply, and the cumulative release peaked at 84%. These results indicate tha when exposed to pepsin in SGF, the nanoparticles remained relatively stable. Howeve in alkaline SIF, a large number of nanoparticles disintegrated, and EGCG was sustainabl released. This can be attributed to the electrostatic interaction between gelatin and PGA which hinders the contact between gelatin and protease. The high cumulative release ra suggests that EGCG/gelatin/γ-PGA nanoparticles serve as superior EGCG delivery carr ers.

DPPH and ABTS Scavenging Activity of Nanoparticles
The antioxidant activity of EGCG/gelatin and EGCG/gelatin/γ-PGA nanoparticles was compared to free EGCG using the DPPH and ABTS radical scavenging assays. As depicted in Figure 5, the antioxidant capacity of all samples increased with the concentration of EGCG, while that of both types of EGCG-embedded nanoparticles decreased slightly with increasing incubation time. In contrast, the antioxidant activity of free EGCG was significantly reduced.
On day 0 of storage, the DPPH radical scavenging activity of free EGCG was 93.3%, while that of EGCG/gelatin and EGCG/gelatin/γ-PGA nanoparticles were 86.3% and 90.7%, respectively ( Figure 5A). As storage time progressed to day 7, the DPPH scavenging rate of free EGCG decreased significantly, falling below that of the two types of nanoparticles ( Figure 5B). By day 14, the DPPH scavenging rate of EGCG/gelatin/γ-PGA nanoparticles was still maintained at 86.2%, which was higher than the 75.6% of EGCG/gelatin nanoparticles and the 39.1% of free EGCG ( Figure 5C). The DPPH scavenging rate of free EGCG decreased by 2.4 times. These results suggest that the nanoparticles might shield EGCG from environmental oxygen oxidation during storage. Moreover, the EGCG/gelatin/γ-PGA nanoparticles had a greater antioxidant capacity than EGCG/gelatin nanoparticles throughout the storage period. This could be attributed to the more compact structure of EGCG/gelatin/γ-PGA nanoparticles, which reduced the contact of EGCG with oxygen.
Similarly, the ABTS radical-scavenging activities of all samples exhibited comparable trends ( Figure 5C-E). After 14 days of storage, the ABTS scavenging rate of EGCG/gelatin/γ-PGA nanoparticles remained at 87.9%, higher than that of EGCG/gelatin nanoparticles (76.2%), while the ABTS scavenging rate of free EGCG decreased to 37.3% ( Figure 5E). These findings imply that the EGCG was nearly fully preserved after being encapsulated in EGCG/gelatin/γ-PGA nanoparticles, indicating the protective effect of the nanoparticles.

DPPH and ABTS Scavenging Activity of Nanoparticles
The antioxidant activity of EGCG/gelatin and EGCG/gelatin/γ-PGA nanoparticle was compared to free EGCG using the DPPH and ABTS radical scavenging assays. A depicted in Figure 5, the antioxidant capacity of all samples increased with the concentra tion of EGCG, while that of both types of EGCG-embedded nanoparticles decrease slightly with increasing incubation time. In contrast, the antioxidant activity of free EGCG was significantly reduced. On day 0 of storage, the DPPH radical scavenging activity of free EGCG was 93.3% while that of EGCG/gelatin and EGCG/gelatin/γ-PGA nanoparticles were 86.3% an 90.7%, respectively ( Figure 5A). As storage time progressed to day 7, the DPPH

EGCG Transport on Caco-2 Monolayers
The oral bioavailability of EGCG is reported to be very low, with only 0.1-1.1% of the administered dose reaching systemic circulation in human studies [46]. Generally, the bioactivity of EGCG is enhanced at concentrations higher than 10 and 20 mol/L. However, due to the significant biotransformation of EGCG in the intestine, the peak plasma concentration of EGCG in the human body is typically around 1 µmol/L [47]. As a result, improving the intestinal absorption of EGCG using nanoparticle carriers is highly significant.
The TEER value is an indicator of the integrity of the Caco-2 monolayers, and a decrease in this value suggests a disruption in the tight monolayer connections. Before the transportation experiment, the Caco-2 monolayers had a suitable TEER value of 400-500 Ω × cm 2 [48]. Figure 6A illustrates the impact of the nanoparticles on TEER during the experiment. The TEER percentage of the control group treated with HBSS buffer decreased to 96.2% of the initial value after 120 min of incubation. The TEER percentages of the Caco-2 monolayers treated with free EGCG, EGCG/gelatin nanoparticles, and EGCG/gelatin/γ-PGA nanoparticles decreased to 93.7%, 81.3%, and 75.8% of the initial value, respectively. The TEER values of the Caco-2 monolayers treated with nanoparticles decreased significantly compared to controls, and the EGCG/gelatin/γ-PGA nanoparticles had a greater impact on the TEER value. It has been found that the paracellular pathway may mediate macromolecular transport in Caco-2 monolayers [49]. When the tight junctions between Caco-2 cells open, molecules pass across the gap between the epithelial cells, causing the TEER value to decrease. Therefore, it appears that the EGCG/gelatin/γ-PGA nanoparticles may promote the penetration of EGCG through the paracellular pathway by opening the tight connections of Caco-2 monolayers. The cumulative transport of EGCG through Caco-2 monolayers was investigated a ter incubation with EGCG, EGCG/gelatin nanoparticles, and EGCG/gelatin/γ-PGA nano particles for up to 120 min ( Figure 6B). The amount of EGCG transported from the dono compartment to the receiver compartment gradually increased with incubation time. A 120 min, the cumulative transport rate with EGCG/gelatin/γ-PGA nanoparticles wa 14.4%, which was higher than the rates of 4.7% for free EGCG and 11.2% for EGCG/gelati nanoparticles. Nanoparticles smaller than 300 nm can pass through the cell membrane vi clathrin-mediated endocytosis, which can increase the permeability of bioactive sub stances [50]. In this study, the permeability of EGCG was enhanced by embedding it i nanoparticles, likely due to the cellular absorption of the nanoparticles by the Caco-2 mon olayers. Furthermore, compared to EGCG/gelatin nanoparticles, the cell transport rate o EGCG in EGCG/gelatin/γ-PGA nanoparticles was higher, possibly due to γ-PGA enhanc ing the cellular absorption of EGCG [23]. Co-administration of EGCG with γ-PGA ha been shown to improve the therapeutic effect of EGCG on metabolism in obese type diabetic mice [51]. Therefore, EGCG/gelatin/γ-PGA nanoparticles may better exert th health properties of EGCG, with potential applications in functional foods.

Conclusions
In this study, we present a novel self-assembly nanotechnology approach to prepar EGCG/gelatin/γ-PGA nanoparticles by simply mixing type-A gelatin with γ-PGA an EGCG in an aqueous solution. The EGCG/gelatin/γ-PGA nanoparticles had larger particl sizes than the EGCG/gelatin nanoparticles. Furthermore, the EGCG/gelatin/γ-PGA nano particles exhibited improved long-term storage stability and antioxidant activity o EGCG. These nanoparticles were found to be stable in SGF and released in SIF. Addition ally, the TEER value and cumulative transport rate indicated that the EGCG/gelatin/γ PGA nanoparticles enhanced the cell absorption of EGCG. Our findings suggest tha EGCG/gelatin/γ-PGA nanoparticles could serve as an effective carrier for improving th stability and bioavailability of EGCG, which could have potential applications in the func tional food industry. The cumulative transport of EGCG through Caco-2 monolayers was investigated after incubation with EGCG, EGCG/gelatin nanoparticles, and EGCG/gelatin/γ-PGA nanoparticles for up to 120 min ( Figure 6B). The amount of EGCG transported from the donor compartment to the receiver compartment gradually increased with incubation time. At 120 min, the cumulative transport rate with EGCG/gelatin/γ-PGA nanoparticles was 14.4%, which was higher than the rates of 4.7% for free EGCG and 11.2% for EGCG/gelatin nanoparticles. Nanoparticles smaller than 300 nm can pass through the cell membrane via clathrin-mediated endocytosis, which can increase the permeability of bioactive substances [50]. In this study, the permeability of EGCG was enhanced by embedding it in nanoparticles, likely due to the cellular absorption of the nanoparticles by the Caco-2 monolayers. Furthermore, compared to EGCG/gelatin nanoparticles, the cell transport rate of EGCG in EGCG/gelatin/γ-PGA nanoparticles was higher, possibly due to γ-PGA enhancing the cellular absorption of EGCG [23]. Co-administration of EGCG with γ-PGA has been shown to improve the therapeutic effect of EGCG on metabolism in obese type 2 diabetic mice [51]. Therefore, EGCG/gelatin/γ-PGA nanoparticles may better exert the health properties of EGCG, with potential applications in functional foods.

Conclusions
In this study, we present a novel self-assembly nanotechnology approach to prepare EGCG/gelatin/γ-PGA nanoparticles by simply mixing type-A gelatin with γ-PGA and EGCG in an aqueous solution. The EGCG/gelatin/γ-PGA nanoparticles had larger particle sizes than the EGCG/gelatin nanoparticles. Furthermore, the EGCG/gelatin/γ-PGA nanoparticles exhibited improved long-term storage stability and antioxidant activity of EGCG. These nanoparticles were found to be stable in SGF and released in SIF. Addition-ally, the TEER value and cumulative transport rate indicated that the EGCG/gelatin/γ-PGA nanoparticles enhanced the cell absorption of EGCG. Our findings suggest that EGCG/gelatin/γ-PGA nanoparticles could serve as an effective carrier for improving the stability and bioavailability of EGCG, which could have potential applications in the functional food industry.

Data Availability Statement:
The data used to support the findings of this study can be made available by the corresponding author upon request.