ε-Polylysine/Sodium Alginate Bilayer-Modified Nanoliposomes Enhancing the Stability and In Vitro Bioavailability of Epigallocatechin Gallate

Abstract

Epigallocatechin gallate (EGCG) represents the key phenolic compound in green tea, which has been verified to possess various biological effects but suffers from low stability and poor bioavailability. To address these issues, EGCG-loaded nanoliposomes (ELs) were screened and prepared using an ethanol injection–calcium acetate gradient (EtOH-CAG) method. An encapsulation efficiency of 94.61% was achieved, involving a particle size of 118.6 nm and a polydispersity index (PDI) of 0.23. Via layer-by-layer assembly, nanoliposomes modified with either ε-polylysine (ε-PL) monolayer (ELP) or ε-polylysine/sodium alginate (SA) bilayer (ELPA) exhibited substantially improved stability. Moreover, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and thermal analysis confirmed the effective loading of EGCG and the successful decoration of ε-PL and SA. Molecular docking analyses of dual ligands further characterized the surface modification mechanism, which was primarily mediated by hydrogen bonding and electrostatic interactions. ELPA maintained robust stability under conditions including 200 mM salt concentration, a pH range of 4–10, temperatures up to 55 °C, and a 25-day storage duration. The modified systems showed considerably enhanced cellular uptake without causing cytotoxicity. Collectively, the developed ε-PL/SA bilayer nanoliposomes offer an eco-friendly, efficient strategy to enhance EGCG stability and in vitro bioavailability in functional food applications.

1. Introduction

Polyphenols, a structurally diverse class of secondary metabolites ubiquitously distributed in plant-derived foods, exhibit multifaceted biological functionalities attributed to their hydroxyl-rich aromatic frameworks [1]. These compounds possess considerable health potential, positioning them as promising functional food ingredients. (-)-Epigallocatechin gallate (EGCG), which is the most abundant polyphenol in green tea and has been the subject of a great deal of research [2,3,4], has been widely employed in the food industry [5]. However, the distinctive architecture of EGCG, particularly its ortho-dihydroxy and vicinal-trihydroxy moieties, confers potent bioactivity but simultaneously acts as a highly unstable structural liability, driving oxidative degradation and epimerization [6,7,8]. Numerous studies have indicated that catechins, particularly EGCG, are highly unstable during storage and processing, with marked susceptibility to oxidation, light exposure, and pH fluctuations. Storing EGCG solutions at concentrations of 0.05 mg/mL and 0.1 mg/mL in sunlight and ambient air results in complete degradation within 48 h [9]. Upon heating to 80 °C, the complete degradation of EGCG can be accelerated to just 25 min [10]. In addition, it has been demonstrated that following 30 min of treatment with phosphate-buffered solution (PBS) at pH 7.4, EGCG content drops to less than 40% and fully degrades after 2 h at 37 °C [11]. The inherent instabilities further contribute to the reduced stability of EGCG within the gastrointestinal tract [12,13]. Furthermore, the limited intestinal permeability associated with partial deviation from Lipinski’s rule of five—an empirical guideline used to predict the oral bioavailability of small molecules based on molecular weight, lipophilicity, and hydrogen-bonding capacity—results in poor absorption and minimal oral bioavailability [14,15,16]. This, in turn, causes inefficient utilization, and significant waste of plant resources. As documented in the existing literature, EGCG exhibits an oral bioavailability of only around 0.1% in humans and experimental animals [17,18,19]. Consequently, there is a pressing need to develop advanced and effective solution strategies regarding its structural properties.

In recent years, nanocarrier-based delivery systems have attracted increasing attention as effective strategies to address the inherent instability and low bioavailability of polyphenolic compounds [20]. Their amphiphilic nature facilitates the co-encapsulation of hydrophilic compounds within the aqueous core and hydrophobic molecules within the lipid bilayer, thereby providing a versatile platform for polyphenol stabilization [21,22]. Additionally, the flexible vesicular structure of nanoliposomes enhances cellular uptake through membrane fusion while promoting transport across biological barriers, rendering nanoliposomes advantageous for oral delivery. Early proof-of-concept work involving tea-derived polyphenols demonstrated that followed by probe sonication, thin-film hydration afforded vesicles with 45% entrapment efficiency [23]. These tea-polyphenol nanoliposomes resisted aggregation at pH 2–7 and showed sustained release over 24 h, establishing the feasibility of loading multicomponent catechin–gallic acid systems. Subsequent studies focused on process intensification. Specifically, Peng and co-workers [24] designed a pH-driven loading protocol, in which alkali-solubilized polyphenols were protonated in situ inside pre-formed vesicles, pushing the encapsulation efficiency above 80% while maintaining mean diameters below 90 nm. Despite a modest increase in lipid oxidation, the strategy markedly improved polyphenol co-delivery in simulated digestion models. More recently, academic interest has shifted to the valorization of plant-derived waste streams. Utilizing ultrasound-assisted extraction, Prevete et al. [25] produced a polyphenol-rich extract from olive leaves and orange peels, which was subsequently encapsulated in soy-lecithin nanoliposomes. This formulation preserved over 90% of the original hydroxytyrosol–hesperidin content after 30 days at 25 °C and potentiated the antimicrobial activity against Listeria monocytogenes. Parallel work in the food sector demonstrated that deoiled-lecithin nanoliposomes could accommodate 26 mg/mL of mixed phenolics from rosemary, improving thermal stability by 3-fold and delaying lipid peroxidation in model emulsions [26].

Multiple effective preparation strategies have been developed for the production of nanoliposomes. Active loading, a remote-loading technique, has demonstrated high encapsulation efficiency (>90%) using food-grade materials without the need for costly equipment or organic solvents. Active loading primarily relies on pH-gradient and ion-gradient strategies, among which the calcium acetate gradient (CAG) method is commonly adopted [27]. The pH-gradient method has recently gained prominence in food applications due to its solvent-free operation and straightforward implementation [28]. However, this approach suffers from critical limitations related to polyphenol encapsulation, particularly its reliance on alkaline conditions and the inherent lability of transmembrane gradients during storage [24,29,30]. The CAG method offers particular promise for weak acidic polyphenols such as EGCG. This synergy is attributable to the inherent physicochemical properties of EGCG, whereby electron delocalization facilitates deprotonation and subsequent formation of stable calcium–phenolate complexes within the liposomal aqueous core [31,32]. Such ion-trapping mechanisms theoretically enable both high encapsulation efficiency and enhanced protection against molecular degradation. However, conventional approaches present substantial hurdles for the fabrication of nanoliposome-based delivery systems, as producing uniformly small particles and attaining high entrapment efficiency remain inherently problematic.

Liposomes, despite their potential in nutrient delivery, are widely acknowledged to be limited by instability due to oxidative and hydrolytic degradation of their phospholipid membranes. This degradation compromises bilayer integrity, leading to premature core material leakage and reduced bioactivity [33]. Chitosan represents a widely used biomaterial in oral delivery systems and plays a crucial role in enhancing liposome stability. However, its preparation involves extended stirring (≥24 h) under strictly controlled acidic conditions and temperatures, which can be time-consuming and inefficient [34]. In contrast, two promising natural biopolymers—ε-polylysine and sodium alginate (SA)—offer advantages such as biocompatibility, biodegradability, and ease of dissolution without the preparation complexities associated with chitosan. Having been granted GRAS (Generally Recognized as Safe) certification by the FDA in 2004 (ε-polylysine) and 2012 (sodium alginate), both compounds are well-positioned for application in biomedical fields [35,36]. Recent studies indicate that liposomes coated with poly-L-lysine and hyaluronic acid are internalized more effectively compared to uncoated liposomes, suggesting that embedded layers enhance liposome–cell interactions rather than hinder them [37]. Furthermore, while limited, several valuable studies have confirmed the low-toxicity and effective multi-liposomal assembly of polylysine and alginate on liposomes. These attempts have determined the interactions between components, their stability in physiological solutions, and their in vitro cytotoxicity profiles [38]. However, further research and validation on their fabrication, interaction mechanisms, stability, safety, and efficacy are required.

This study reported the development and screening of a novel food-grade approach for preparing EGCG nanoliposomes using the ethanol injection–calcium acetate gradient (EtOH-CAG) method. To enhance the efficiency of the nanodelivery system, the liposomes were modified layer by layer with ε-polylysine and sodium alginate. The structure of the liposomes, both pre- and post-modification, was characterized using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). Additionally, dual-ligand molecular docking technology was applied for the first time to visualize and explore the complex principles underlying the layer-by-layer modification of nanoliposomes by biopolymers. The physical stability of the liposomes was subsequently assessed upon exposure to environmental stresses typical of food matrices, including ionic strength, pH variations, NaCl concentration, and heat treatment. Ultimately, the cellular uptake efficiency of the formulated nanoliposomes was compared to determine their relative effectiveness in improving the in vitro bioavailability of EGCG.

2. Materials and Methods

2.1. Materials

Epigallocatechin gallate (EGCG, ≥95%) was purchased from Manster Biotechnology Co., Ltd. (Chengdu, China). Three additional reagents, including lecithin from soybean (≥90%), ε-Polylysine (ε-PL, >95%), and sodium alginate (SA, 200 ± 20 mPa.s), were sourced from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Cholesterol (≥97%) was obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Calcium acetate was purchased from Tianjin Kemio Chemical Reagent Co., Ltd. (Tianjin, China). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen and Gibco, respectively, and 3-(4,5-dimthyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Solarbio (Beijing, China). Other reagents, of either analytical or chromatographic purity, were obtained from Shandong Yuwang Hetianxia New Material Co., Ltd. (Dezhou, China).

2.2. Cell Culture and Conditions

Herein, the rat hepatocyte BRL3A cells were cultured in DMEM containing 10% FBS and antibiotics, maintained at 37 °C in a 5% CO₂ incubator. Cells were grown until they reached 80% confluency.

2.3. Preparation of Nanoliposomes

2.3.1. Preparation of Blank Nanoliposome

The nanoliposomes were produced using the ethanol injection–calcium acetate gradient method (EtOH-CAG) previously described by Katja Istenič et al. [39], with slight alterations. Briefly, a liposome lipid composition of soybean lecithin/cholesterol with a mass ratio of 4:1 was selected. For the preparation of the liposomes, lipids were first solubilized in absolute ethanol and then swiftly injected into calcium acetate solution (125 mmol/L) via a syringe pump at 55 °C. A series of lipid-to-aqueous phase volume ratios (1:6, 1:7, 1:8, 1:9, 1:10, v/v) were tested to screen for the most suitable formulation parameters. The mixture was transferred into a dialysis bag (cut-off Mw 8–14 kDa) after ultrasound for 10 min. Dialysis was carried out using distilled water as the dialysis medium at room temperature under gentle agitation for a total duration of 4 h, with the dialysate refreshed hourly. All samples were passed through 0.22 μm syringe filters. The pH of the resulting solutions was adjusted to 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 for subsequent analyses, and then blank nanoliposomes (BLs) were obtained according to the method described previously by Cern et al. [40].

2.3.2. Remote Loading of EGCG

EGCG was dissolved in water at different molar ratios of 1:4, 1:6, 1:8, 1:10, and 1:12 to lecithin, and then dropped into BL with a syringe. The incubation temperature (30, 35, 40, 45, and 50 °C) and oscillation time (8, 10, 12, 14, and 16 min) were determined with EGCG encapsulation efficiency as evaluation indexes. EGCG nanoliposome (EL) was obtained based on the remote loading method after being filtered through a 0.22 μm Luer Syringe Filter membrane [39].

2.3.3. SA/ε-PL-Modified EGCG Nanoliposome

ε-PL aqueous solution (0.3 mg/mL) was prepared at a lecithin-to-ε-PL mass ratio of 20:1 (w/w). The solution was passed through a 0.22 μm filter membrane and then added dropwise to the EL suspension at 45 °C with magnetic stirring for 30 min, and ε-PL monolayer-modified EGCG nanoliposomes (ELPs) were prepared. For the preparation of SA/ε-PL bilayer-modified EGCG nanoliposomes (ELPAs), SA (0.2 mg/mL) was dissolved in water and then added dropwise to an equal volume of ELP suspension with a magnetic stirrer at room temperature [41]. ε-PL- and SA bilayer-modified EGCG nanoliposomes (ELPAs) were acquired immediately. The above samples, including EL, ELP, and ELPA, were prepared for particle size analysis and morphological observation. All samples were then lyophilized in the presence of cryoprotectant for subsequent testing. For the preparation of blank control samples (BL, BLP, BLPA), all steps were performed in the same manner as described above, with EGCG excluded from the formulation components. For the cellular uptake assay, a hydrophilic fluorescent component (FITC) was incorporated in the nanoliposome synthesis instead of EGCG (FL, FLP, FLPA).

2.3.4. Determination of Encapsulation Efficiency of EGCG

Free EGCG was removed from the liposome system by ultrafiltration [42] with some modifications to determine the actual amount of encapsulated EGCG. The nanoliposome suspension was added to the inner tube within the ultrafiltration tube (100 kDa, Millipore, St. Louis, MO, USA) and centrifuged for 8 min at 3000 g. The free EGCG was obtained from the outer tube. Meanwhile, total EGCG was obtained by dissolving the nanoliposome suspension in 5 times the volume of ethanol for ultrasonic demulsification. The EE% of EGCG was calculated using the following equation:

EE% = (EGCG_Total − EGCG_Free)/(EGCG_Total) × 100

(1)

where EE%—encapsulation efficiency; EGCG_Free—free EGCG concentration; EGCG_total—total EGCG concentration.

The free and total EGCG concentrations were determined by Acquity UPLC coupled with a PDA Detector (Waters, Milford, MA, USA). The sample was filtered through a 0.22 μm syringe filter (Yuwang, Shenyang, China) and injected (0.2 μL) into an Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) at 10 °C through a mobile phase consisting of acetonitrile (containing 0.1% formic acid, solvent A) and water (containing 0.1% formic acid, solvent B). A gradient program with the following profile was adopted: 0–2 min, 7% A; 2–8 min, from 7% to 10% A; 8–9 min, from 10% to 18% A; 9–9.3 min, from 18% to 32% A; 9.3–11.3 min, from 32% to 100% A; and 11.3–13 min, from 100% to 7% A. The flow rate was 0.27 mL/min, and the PDA detection wavelength was set at 280 nm.

2.4. Characterization of Nanoliposomes

2.4.1. Particle Size Analysis and Morphological Observation

The particle size distribution and polydispersity index (PDI) of EGCG nanoliposome systems were measured at 25 °C using a particle size and ζ-potential analyzer (NS-90Z, OMEC, Zhuhai, China). The morphology of EGCG nanoliposome systems was evaluated utilizing transmission electron microscopy (TEM) (HT7800, Hitachi, Tokyo, Japan). In brief, droplets containing freshly prepared EL, ELP, and ELPA were placed on the copper grid, and excess liquid was removed using filter paper after 10 min, respectively. Samples were air-dried, 20 μL of 2% (w/w) phosphotungstic acid was added dropwise for staining for 2 min, and excess dye solution was removed using filter paper. Following natural drying, observation of the morphology of EGCG nanoliposome systems was performed at 100 kV.

2.4.2. Fourier Transform Infrared (FTIR) Spectroscopy

Fourier transform infrared spectroscopy (FTIR-650, Gangdong, Tianjin, China) was utilized to characterize the chemical groups of the nanoliposomes, with measurements carried out at wavelengths of 4000–500 cm⁻¹ with a resolution of 2 cm⁻¹ and 8 scans. Samples were prepared by blending with dry KBr powder, triturating to a fine powder, and pressing into KBr discs via manual pressure for FTIR testing.

2.4.3. X-Ray Diffraction (XRD) Analysis

The crystallinity of samples was recorded using an X-ray diffractometer (Ultima IV, Rigaku, Tokyo, Japan). Data were collected over an 2θ range from 5° to 90° in continuous mode with a speed angle of 5°/min.

2.4.4. Thermal Analysis

Thermal analysis was performed using a Netzsch STA 449 F3 (Netzsch, Selb, Germany). Briefly, approximately 5 mg of lyophilized powder (ε-PL, ALG, CS, CS-ALG NPs and ε-PL NPs) was analyzed using simultaneous thermal analyses (STA) in the analyzer, heated from 30 to 500 °C at a constant heating rate of 20 °C/min under constant purging of nitrogen.

2.4.5. Molecular Docking

ε-PL represents a homopolymer consisting of 25−35 L-lysine monomers. Herein, the structure of ε-PL was generated by Phyre2, a set of web-accessible tools for protein structure modeling, prediction, and analysis [43]. Sodium alginate (SA) is a linear polysaccharide composed of (1 → 4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. To obtain a computationally manageable yet representative model of the polymer, the G and M disaccharide—the minimal repeating motif of SA—was hereby extracted and used in the molecular docking simulations. Lecithin was chosen as a representative of nanoliposome, as it was mainly a phospholipid bilayer structure composed of lecithin. ChemDraw 20.0 and Chem3D 20.0.0.41 were employed to draw and optimize the structures of lecithin, ε-PL, and SA. AutoDockTools 1.5.6 and Autodock vina [44,45] were executed to simulate the receptor–ligand interactions, with ε-PL being set as the receptor and SA combined with lecithin as the dual ligands. Semi-flexible docking was applied to identify the optimal protein–ligand complex structure, in which the protein was regarded as a rigid body, and all the rotatable bonds in ligands were sampled. The grid center was set at the active site gorge (−0.078, 1.568, 1.156), and the grid size axis was set to X = 36, Y = 22.5, and Z = 32.25 with a grid spacing of 0.375. The binding affinity and interactions were visualized in PyMOL 2.6.0a0.

2.5. Stability Evaluation of EGCG-Encapsulated Systems

2.5.1. Storage Stability Assay

The storage stability of EL, ELP, and ELPA was evaluated by analyzing the degradation of the liposomes at 4 °C. Specifically, the present study compared the degradation of EGCG in nanoliposomes during 25 days of storage. The samples were collected every 5 days, and retention rates were recorded via ultra-performance liquid chromatography (UPLC, Waters ACQUITY H–Class) [46].

2.5.2. Evaluation of In Vitro Food Simulation Stability

Hydrodynamic diameter and PDI were used as key indicators to evaluate the stability of lyophilized nanoliposome formulations under variable NaCl concentrations, metal ion types, pH levels, and temperatures. All determinations were carried out on an OMEC NS-90Z instrument, with the procedure modified slightly from that established by Paulina Cerda-Opazo [47]. NaCl concentration in the medium was set to 0–200 mM, and various metal ions, including 5mM Fe²⁺, Fe³⁺, and Mg²⁺, were used as daily dietary environments to preliminarily assess the stability of nanoliposomes, respectively. To alter the pH levels, solutions of HCl (0.01 M) and NaOH (0.01 M) were used to modify pH of the system. In the temperature evaluation experiment, samples were placed in a thermostat water bath and allowed to reach thermal equilibrium for 15 min before each measurement of particle size and PDI.

2.6. Cellular Experimentation

2.6.1. Cell Viability

The MTT assay, as described before, was utilized to evaluate cell viability [48]. Briefly, BRL-3A cells (3 × 10⁴/well) were seeded in 96-well plates and incubated for 24 h in culture medium. Then, cells were exposed to either EGCG-encapsulated nanoliposomes (at EGCG concentrations of 25, 50, and 75 μM) or blank nanoliposome controls for 24 h. Free EGCG was used as a control. Upon treatment, cells were washed twice with PBS and incubated with MTT (0.5 mg/mL) in culture medium at 37 °C for 4 h. Formazan crystals were dissolved in 100 μL of DMSO, and absorbance was measured at 490 nm using a VersaMax microplate reader (St. Louis, MO, USA).

2.6.2. Cellular Uptake

In order to examine the biological functions of the formulations, cellular uptake assays were conducted. BRL-3A cells, at a density of 1 × 10⁴ cells per well, were seeded into 24-well plates containing sterile 14 mm round glass coverslips and cultured for 24 h in a complete growth medium. Following the incubation period, cells were treated with 500 μL of FITC-labeled nanoliposomes (green staining) at 37 °C, and then washed with ice-cold PBS and fixed with 4% paraformaldehyde for 20 min after 2 and 6 h of incubation. Subsequently, the nuclei were stained with Hoechst 33342, resulting in blue fluorescence. The coverslips were then mounted onto glass slides using an antifade mounting medium and examined under a fluorescent microscope (TI-S, Nikon, Tokyo, Japan) [47,49,50].

To determine the average fluorescence intensity, similarly, the BRL-3A cells were seeded in 6-well plates and incubated at 37 °C for 24 h, which were then rinsed with cold PBS and resuspended in 0.5 mL of PBS after 2 and 6 h of FITC-labeled nanoliposome incubation for further analysis using flow cytometry (BD FACSAria, Los Angeles, CA, USA).

2.7. Statistical Analysis

The data obtained based on the physicochemical parameters and cell viability assay of nano-formulations were presented as the mean ± standard error of the mean. These data were derived from three independent experiments conducted in triplicate and analyzed using GraphPad Prism 6.0 software. Statistical analysis for EE%, particle size, and PDI of nanoliposome systems was performed using Tukey’s test as a post hoc test. Statistical analysis for cell viability (untreated control against EGCG, EGCG or blank nanoliposomes treatment) was performed by one-way ANOVA, followed by Dunnett’s post hoc test. A p-value less than 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Parameter Selection and Preparation of Nanoliposomes

In this study, the stable liposomal formulations of EGCG were screened. Taking encapsulation efficiency as the index, different preparation conditions for lipid systems, including various volume ratios of lipid phase to aqueous phase, different pH, differing molar ratios of core material to lipid, incubation times, and incubation temperatures, were investigated to identify the preferred conditions. The results are shown in Figure 1. The highest encapsulation efficiency of EGCG was achieved at a lipid–ethanol phase to calcium acetate aqueous phase volume ratio of 1:8 (Figure 1A). This was attributed to there being a sufficient calcium acetate concentration (125 mM) required to create a driving force for EGCG influx (Figure 2A). However, as the aqueous phase volume further increased to 1:9 and 1:10, the encapsulation efficiency decreased gradually. This could potentially be attributed to the liposome microspheres relying on osmotic pressure differences between the inner and outer aqueous phases for active ingredient loading. Excessive outer aqueous phase resulted in greater EGCG deprivation, consequently decreasing the encapsulation efficiency. Regarding incubation pH, an indispensable parameter for the active loading process, the relevant experimental findings are displayed in Figure 1B. Encapsulation efficiency was assayed at pH values ranging from 4.5 to 7 within the loading system. The results revealed a trend of an initial slight increase followed by a decrease. Encapsulation efficiency peaked at pH 5.5 and was lowest at pH 7, consistent with the instability of EGCG under non-acidic conditions [11]. Different molar ratios of EGCG to lecithin could significantly affect the encapsulation efficiency, as shown in Figure 1C. The highest encapsulation efficiency was obtained at an EGCG to lipid molar ratio of 1:4.

The incubation time and temperature for remote loading phytochemicals are often important factors that affect encapsulation efficiency. As shown in Figure 1D,E, the incubation at 45 °C for 10 min was observed to be the selected condition for EGCG encapsulation, under which the encapsulation efficiency reached 94.61 ± 0.02%.

3.2. Characterization of SA/ε-PL Coating of EGCG Nanoliposomes

3.2.1. Encapsulation Efficiency, Particle Size and PDI

To further enhance the properties of the liposome system, ε-PL and SA were introduced to modify the nanoliposomes. ε-PL monolayer-modified ELP and ε-PL and SA bilayer-modified ELPA were fabricated. The encapsulation efficiency, particle size, and PDI of the prepared nanoliposome systems were determined and summarized, as shown in

Table 1. EE%, particle size, and PDI of nanoliposome systems loaded with EGCG.

Nanoliposomes	EE%	Particle Size (nm)	PDI
BL	——	112.6 ± 2.8 a	0.19 ± 0.01 ac
EL	94.61 ± 0.02 a	118.6 ± 5.2 b	0.23 ± 0.02 b
ELP	95.35 ± 0.09 a	119.9 ± 5.1 b	0.19 ± 0.03 ab
ELPA	95.73 ± 0.14 a	120.9 ± 5.8 b	0.18 ± 0.02 c

Note: Results were expressed as the mean ± SD. Statistical analysis was performed using Tukey’s test as a post hoc test. Different lowercase letters within the same column indicate significant differences (p < 0.05). EE%: encapsulation efficiency; PDI: polydispersity index; BL: blank nanoliposome; EL: EGCG-loaded nanoliposomes; ELP: ε-polylysine monolayer-modified nanoliposomes; ELPA: ε-polylysine/sodium alginate bilayer-modified nanoliposomes.

. The encapsulation efficiency values of EL, ELP, and ELPA were 94.61 ± 0.02%, 95.35 ± 0.09%, and 95.73 ± 0.14%, respectively. This suggested that the absolute encapsulation efficiency values were numerically increased after ε-PL and SA modifications; however, these differences were not statistically significant. These findings demonstrated superior performance compared to previously reported EGCG delivery systems. Jara-Quijada et al. [51] prepared chitosan-coated liposomes loaded with green tea polyphenols using the ethanol injection method followed by electrostatic deposition of chitosan. However, this approach achieved only 61.31% encapsulation efficiency, which was significantly lower than that of the ELPA system. This discrepancy might be attributed to the passive loading approach, which typically resulted in limited entrapment and potential leakage during the preparation process. In contrast, Istenič et al. [39] developed a liposome–chitosan and alginate microparticles composite system using the ethanol injection method combined with spray-drying, which achieved an encapsulation efficiency exceeding 97% EGCG. However, this system exhibited a broad particle size distribution in the micrometer range and presented challenges with regard to re-dispersibility, limiting its practical applications in aqueous food systems.

The average particle sizes of the BL, EL, ELP, and ELPA were 112.3 ± 2.8 nm, 118.6 ± 5.2 nm, 119.9 ± 5.1 nm, and 120.9 ± 5.8 nm, respectively. These results indicated that the average sizes of all formulations ranged between 100 and 150 nm and were therefore appropriate for high-efficiency delivery via cell fusion and endocytosis [52]. Meanwhile, the dispersion of ELPA was slightly improved, with the PDI decreasing significantly from 0.23 to 0.18. As described above, the ethanol injection method produced formulations with particle sizes in the hundreds of nanometers [39,51]. By contrast, in the two studies on pectin–chitosan nanoliposomes, Shishir et al. [53,54] prepared nanocarriers via the thin-film hydration method followed by layer-by-layer assembly, with particle sizes ranging from 87.1 to 444.77 nm and PDIs between 0.14 and 0.40. Although their bilayer polysaccharide modification strategy shared conceptual similarities with the layer-by-layer assembly approach employed in the ELPA system, a considerably broader size distribution was observed. This might have adversely affected the physicochemical stability and sensory properties of the final product [52].

Notably, protein-based delivery systems have garnered increasing attention with regard to tea polyphenols. The β-lactoglobulin–gum Arabic complex nanoparticles fabricated by Gao et al. [55] achieved a remarkably high encapsulation efficiency of 84.5%. Similarly, Xie et al. [56] fabricated Zein–Lecithin–EGCG complex nanoparticles using the antisolvent coprecipitation method, achieving a particle size of 221.0 nm with an encapsulation efficiency of 68.5%. This discrepancy could be attributed to the passive encapsulation mechanism relying primarily on hydrophobic interactions and hydrogen bonding between proteins and EGCG. Such interactions offer limited driving force for loading compared to the transmembrane ion gradient employed in the liposomal active loading strategy. Taken together, these findings indicated that the bilayer modification produced a balanced improvement in physical properties, with a particularly clear improvement in system homogeneity, while maintaining comparable encapsulation efficiency and particle size.

Figure 2B presents a schematic of the layer-by-layer assembly of ε-PL and SA on EGCG nanoliposomes prepared using the EtOH-CAG method. The ε-PL (positive charge) and SA (negative charge) were modified onto the surface of liposomes (positive charge) via electrostatic interactions. Evidence showed that the ε-PL and SA-mediated coating was more stable compared to the direct insertion of cholesterol into the lipid bilayer [57]. Relative to EL, the particle size of the nanoliposomes increased with the layered modifications of PL and SA, possibly due to the protein–polysaccharide complex acting as a physical barrier on the nanoparticle surface [58]. Although there was a slight increase in particle size, all formulations remained within the nanoscale range necessary for high-efficiency delivery via cell fusion and endocytosis [52]. This was considered acceptable for use as a lipid-based delivery system [52]. The prepared formulation maintained a consistently high encapsulation efficiency following SA encapsulation. This could be due to the electrostatic deposition of biopolymers on lipid bilayers forming a dense polymer shell, which reduced membrane permeability and limited EGCG diffusion [36,59,60,61]. With a measured PDI value of 0.18 for ELPA, the resultant nanoliposomes exhibited a highly restricted size distribution compared to EL, forming a uniform lipid-based formulation assembly [62].

3.2.2. Morphological Analysis

Figure 3A shows TEM microphotographs where nanoliposomes stained with phosphotungstic acid appear darker at the aqueous core, while the surrounding lighter area is the phospholipid bilayer [63]. The TEM image demonstrates that the prepared formulations exhibited a predominantly spherical shape and uniformly distributed particle size of around 120 nm, which were consistent with the corresponding particle size and PDI measurements. The particles had the dimpled surface characteristic of negatively stained liposomes, confirming the integrity of the vesicular structure [42,62,64]. As observed in Figure 3A(iii), the SA modification concealed the phospholipid bilayer structure of the liposomes. By forming a protein–polysaccharide complex through electrostatic deposition, the formulation became increasingly uniform and dispersed, which enhanced colloidal stability and facilitated controlled release of the encapsulated elements [58].

3.2.3. FTIR Analysis

FTIR was employed to confirm the encapsulation and possible interactions of EGCG with the polymer nanoliposome formulations. The results are shown in Figure 3B. The infrared spectrum of EGCG displayed notable peaks: a broad peak around 3500 cm⁻¹ from phenolic hydroxyl group stretching, and peaks at 1532, 1515, and 1695 cm⁻¹ from the aromatic ring and gallic acid carbonyl stretching. In the FTIR spectrum of the blank liposome, small peaks at 2928 cm⁻¹, 2856 cm⁻¹, and 670 cm⁻¹ corresponded to the antisymmetric CH₂ stretching, symmetric CH₂ stretching, and CH₂ wagging vibrations of the acyl chains, respectively. The peak near 1420 cm⁻¹ corresponded to CH₂ scissoring, while that at 1738 cm⁻¹ corresponded to carbonyl stretching vibration of the lecithin. Peaks near 1090 cm⁻¹ and 1240 cm⁻¹ corresponded to symmetric and antisymmetric PO₂⁻ stretching vibration, respectively. In the ε-PL spectrum, two shoulders were found near 1661 cm⁻¹ and a peak at 1255 cm⁻¹, which could be assigned to carbonyl stretching vibration, N-H deformation vibration, and C-N bond vibration. They corresponded to the strong amide I, II, and III absorbance bands of ε-PL, respectively. The FTIR characteristic absorption bands of SA were observed at 1597 cm⁻¹, and were indicative of the typical COO⁻ stretching vibration of carboxylate salt group, and the absorption peaks near 1412 cm⁻¹ were attributed to the deformation and bending vibration of CH₂ and CH. In addition, the absorption peaks at 1200−1000 cm⁻¹ in the infrared spectrogram of SA were attributed to the C-O stretching vibration peak of pyranose ring in the polysaccharide [41,65]. By comparison, the FTIR spectra of the measured raw materials were basically consistent with those in the literature.

Characteristic peaks of EGCG disappeared in both uncoated and ε-PL/SA-coated nanoliposomes, which indicated that EGCG had been successfully encapsulated within the inner aqueous core of the nanoliposome formulations. In addition, the FTIR spectra of ELP and ELPA showed characteristic peaks for ε-PL and SA at 1417 cm⁻¹ and 1151 cm⁻¹ after modification, compared to EL and ELP, respectively. For instance, the FTIR evidence suggested that the carbonyl peak of BL shifted from 1738 cm⁻¹ to 1736 cm⁻¹ upon ε-PL modification. Upon further binding of SA, a slight red shift to 1737 cm⁻¹ occurred (ELPA), which might have been related to the spatial site resistance of SA and the formation of new hydrogen bonds. Moreover, the electronic interaction between positively and negatively charged macromolecules was evidenced by the shifts from 1240 cm⁻¹ (BL) to 1237 cm⁻¹ (ELP) and from 1451 cm⁻¹ (ELP) to 1445 cm⁻¹ (ELPA). Such shifts were attributed to the attraction between amino and phosphate or carboxyl oxygen atoms [39,41,65,66]. These FTIR results further confirmed the successful preparation of bilayer-modified nanoliposomes.

3.2.4. XRD Analysis

As shown in Figure 3C, free EGCG exhibited significant crystalline peaks at 13.78, 17.04, 17.52, 21, 23.42, 26.32, 28.88, 31.38, and 38.94°, while none were observed in EL, ELP, and ELPA in the XRD patterns [67,68]. This demonstrated the amorphization of EGCG in the nanoliposomes, indicating the successful encapsulation of EGCG. The spectra of EL resembled those of the BL, showing the typical pattern of lipids, characterized by a weak diffused halo centered at 20° 2 θ and several narrow peaks at 5.22, 7.4, 10.18, 10.52, 11.78, and 15.82° 2 θ. They were also accompanied by a series of sharp small peaks around 26° 2θ [69,70]. According to previous studies, major characteristic diffraction peaks without long-range order of crystalline materials of ε-PL and SA appeared in the pattern at around 23.61° and 13.62° [71]. However, after ε-PL and SA modification, the ELP and ELPA patterns almost had no visible peaks compared to EL. These remarkable changes might illustrate that adding ε-PL and SA induced the formation of amorphous nanostructures [67,72], which was mainly due to the formation of hydrogen bonds between polymers [73]. The amorphous form was considered to have higher energy content, an enhanced surface area, and, consequently, increased solubility, dissolution rate, and bioavailability [74]. XRD patterns showed that ε-PL and SA effectively modified the liposomes, promoting a shift to an amorphous state, potentially increasing bioavailability.

3.2.5. Thermodynamic Properties

The DSC curves are displayed in Figure 3D(i); free EGCG exhibited a sharp endothermic peak at 255 °C, which corresponded to its melting point, consistent with the thermodynamic characteristics of crystalline EGCG. The first endothermic peak of BL appeared at 81.3 °C, indicating that the phospholipid bilayer transformed to the fluid liquid crystalline phase [42,75]. The thermogram of EL, ELP, and ELPA revealed that the absence of the characteristic endothermic peaks at 255.26 °C for EGCG indicated the transition to an amorphous form, simultaneously confirming the effective interaction with lipids. According to a previous report, switching from the crystalline to the amorphous phase improved entrapped substances’ saturation solubility and prevented Ostwald ripening [76]. This matched the XRD outcomes, which pointed to an enhancement in stability. Slight shifts near 80 °C of the peaks of EL, ELP, and ELPA were probably attributed to changes in the microstructure of the liposome bilayer membranes caused by interactions between lecithin and other molecules. Moreover, neither the characteristic peak of ε-PL nor the certain endothermic peak at 84.24 °C and exothermic peak near 250 °C of SA were observed in the DSC curves. However, following ε-PL and SA modification, some effects were observed which were opposite to the effects of EL [39,77]. This suggested that the composite system had developed new interactions instead of just being a physical mixture. While the peaks of the EGCG in the DSC curves were not apparent due to the trace amounts, the TG and DTG curves revealed a notable difference in the thermodynamic trend of EGCG between the PM group and the nanoliposome groups. The results demonstrated that the successful modification of the liposome surface with polyelectrolytes based on interactions was consistent with the corresponding functional group changes observed in the FTIR spectra [39,66].

3.2.6. Molecular Docking Results

Figure 4A presents a molecular docking surface diagram of ε-PL and SA bilayer-modified nanoliposomes, where the gray part represents ε-PL as the acceptor, while the pink and orange structures represent SA and lecithin as the dual ligands, respectively.

More interaction details are shown in Figure 4B,C, where the helical structure stands for ε-PL. As shown in Figure 4B, the interaction was mainly focused on the polar head of lecithin with ε-PL, involving several hydrogen bonding and charge interactions. The positively charged amino group at the end of the L-lysine alkane chain generated a mutually attractive charge interaction with the negatively charged phosphate group in the lecithin, while also establishing a hydrogen bond with the oxygen atom in the phosphocholine. Furthermore, according to the optimal conformation, the carbonyl oxygen on the saturated fatty acid chain and the ester oxygen on the unsaturated fatty acid side of lecithin each formed intermolecular hydrogen bonds with the amino groups of the L-lysine side chains. The distances of these interacting bonds all fell in the range of 3.0−3.5. With respect to the interaction of SA with ε-PL (Figure 4C), we observed the charge attraction between the -COO⁻ of gulono- and mannuronic acid with the amino group of ε-PL, as well as hydrogen bonding interactions. In addition, the oxygen within the pyranose ring of SA also led to the formation of a hydrogen bond with the amino group of ε-PL with a distance of 3.2. Moreover, the binding affinity of SA and lecithin with ε-PL was determined to be −4.911 kcal/mol using Autodock vina, which was well above the van der Waals forces. All of these factors could reduce the system’s energy, thereby promoting the development of stable complexes [78]. These comprehensive analyses offered visualized insights into the molecular mechanisms, which were consistent with the FTIR results and provided qualitative support for the proposed layer-by-layer assembly strategy. It should be noted, however, that due to the structural simplifications inherent in the docking approach, these computational results serve as supportive evidence rather than definitive mechanistic validation.

3.3. Stability of EGCG-Encapsulated Systems

3.3.1. Storage Stability

Figure 5 presents the changes in EGCG retention rates for EL, ELP, and ELPA during 25 days at 4 °C. Obviously, EGCG exhibited more rapid leakage in EL and ELP compared to ELPA. The EGCG retention rates for EL and ELP were 80% and 82%, respectively, while ELPA maintained 90% retention by the 25th day of storage. In addition, the EGCG retention rate of ELPA remained higher than that of EL throughout the storage period, followed by ELP. These results confirmed the efficacy of ε-PL and SA bilayer-modified nanoliposomes in strengthening stability. This might be attributed to the adsorption of polymeric compounds on the liposome surface forming a protective barrier, as shown in Figure 4, which reduced EGCG release and consequently enhanced storage stability [79,80].

3.3.2. In Vitro Food Simulation Stability

EGCG-encapsulated systems were examined following exposure to various salt concentrations, metal ions, and pH levels, with the results presented in Figure 6A–C, respectively. It should be noted that different temperatures during storage, transportation, and ingestion could significantly impact the stability of the colloidal system (Figure 6D).

As shown in Figure 6A, the hydrodynamic diameter and PDI of bilayer ELPA remained statistically stable when the NaCl concentration was increased from 0 to 200 mM, indicating that none of the formulations underwent measurable aggregation or fusion under the tested ionic strengths. Previous studies showed that food-grade liposomal systems initiated aggregation when exposed to high NaCl concentrations, as charge screening diminished the electrostatic repulsion between vesicles [47,81,82]. When chitosan or pectin was used as a single coating layer, particle growth of 20–30% was observed at as little as 150 mM [83]. In contrast, the present bilayer ELPA formulation preserved both size and colloidal clarity across the full 0–200 mM range, which could be ascribed to the inherent cohesion of Ca²⁺-stabilized lipid bilayers generated by the calcium acetate gradient and also the electro-steric plus steric hindrance stabilization driven by ε-PL and SA [79,84,85]. Overall, these data underlined the superiority of the EtOH-CAG/ layer-by-layer strategy for applications in high-salt food matrices without loss of colloidal integrity or risk of premature payload leakage. This was essential for ensuring the stability of the dispersion and sustaining the desirable sensory attributes of salt-rich products, including soups, sauces, and sports drinks [86].

Simulated stability experiments on food matrices were conducted in the presence of 5mM Fe³⁺, Fe²⁺, and Mg²⁺ irons, respectively, with the results presented in Figure 6B. In contrast to the limited effect of monovalent cations on liposomes shown in Figure 6A, multivalent metal cations were obviously observed to exert a stronger impact, consistent with studies conducted by Yu et al. [87]. For trivalent iron ions, limited effects on the particle size and PDI of ELP and ELPA were observed. EL, however, exhibited an increased particle size, as shown in Figure 6B(i). Research indicated that trivalent metal ions that are in direct contact with the interfacial region of liposomes could induce gelation by removing water molecules from the lipid vesicles [88,89]. This demonstrated the validity of bilayer-modified liposomes in simulating food environments [87]. In the present work, as depicted in Figure 6B(ii), the presence of divalent iron ions dramatically increased the EL particle size, with PDI exceeding 0.8. This indicated that homeostatic dysregulation might be caused by the Fenton reaction, as observed by Ou et al. [90]. In comparison to EL, the modified nanoliposomes showed an increased particle size, which was attributable to the adsorption and concurrence of ionotropic gelation when ferrous ions were present [91,92]. Despite this increase, the PDI remained under 0.3, suggesting that the colloidal system was stably dispersed [62]. Similar results were observed in the presence of magnesium ions, as demonstrated in Figure 6B(iii). SA modification effectively maintained the stability of EGCG liposomes, while EL showed an increase. Research indicated that the larger hydration radius and electronegativity of magnesium ions exerted a strong effect on the membrane fluidity of fully saturated phosphatidylserine, leading to increased membrane rigidity-induced leakage and particle size [93]. The dual-layer modification strategy enhanced the resilience of liposomes to complex matrices containing millimolar concentrations of metal ions, such as mineral-fortified products; otherwise, the adverse aggregation and leakage would reduce the shelf life and compromise the delivery of nutrients in fortified foods [94].

Figure 6C presents the stability of EL, ELP, and ELPA at various pH conditions. ELPA maintained a nearly constant particle size and PDI from pH 4 to 10, indicating excellent structural stability in both acidic and alkaline environments. In contrast, monolayer ELP and unmodified EL merely remained stable at pH 4–7. When pH > 7, the particle size and PDI increased significantly, with the EL exceeding 250 nm. This implied disruption to the effective operational particle size range of the nanoliposomes, resulting in an ineffective application. The pH stability of liposomes was critical for incorporating these particles into foods with different acidities and ensuring survival under the pH fluctuations experienced during gastrointestinal passage [95]. It was reported that sodium alginate formed hydrogel layers on the surface of liposomes through cross-linking, creating steric hindrance effects that improved the tolerability of liposomes [79]. The results obtained in the present work confirmed that the double-layer biopolymer modification strategy enhanced the stability of nanoliposomes, particularly under alkaline conditions. This versatility expanded potential applications from acidic fruit juices and fermented dairy products, through neutral milk emulsions, to alkaline food matrices such as egg noodles and baked goods.

Figure 6D shows that the liposomes maintained favorable stability between 30 and 55 °C, regardless of the biopolymer modification. This finding validated their practical applicability under mild thermal processing conditions or elevated temperatures encountered during distribution and storage. ε-PL exhibited strong electrostatic adsorption onto the anionic phospholipid surface, leading to the formation of a dense polycationic coating. This coating increased bilayer rigidity and prevented heat-induced membrane fluidization, keeping the liposomal size and PDI stable up to 55 °C [85]. Adding a hydrated sodium-alginate shell created an external barrier that reduced vesicle collisions and lipid exchange, preserving the nanoscale size while maintaining low dispersity under thermal stress [96]. This layer-by-layer assembly of ε-PL and SA provided dual stabilization, similar to the thermal stability of alginate-sheathed liposomes with cinnamon essential oil [97]. ELPA’s stability across diverse conditions could establish it as a promising and viable candidate for use as a food ingredient.

3.4. Evaluation of Cytotoxicity and Cellular Uptake by BRL-3A Cells

Figure 7 presents the results of the cytotoxicity of the formulations at various concentrations via an MTT assay. As can be seen in Figure 7A, when the nanoliposome formulation was incubated at a concentration of 75 μM, no significant variation in cell viability was observed compared to normal hepatocytes. These results indicated the non-cytotoxic nature and excellent biocompatibility of the nano-formulation, as expected [79,98,99].

The cellular uptake of FITC-labeled liposomes was observed using an inverted fluorescence microscope (Figure 7B). There were no significant changes in cell morphology throughout the incubation period. Following the 2 h incubation period, yellow-green fluorescence appeared in the cytoplasm of BRL-3A cells, indicating successful liposome internalization. FLPA exhibited slightly weaker fluorescence compared to FL, followed by FLP. This could be attributed to the sustained-release properties upon SA modification.

Nevertheless, as shown in Figure 7B(ii), after 6 h of incubation, a pronounced increase in fluorescence intensity with more uniform distribution was observed across all groups. Compared to unmodified nanoliposomes, the double layer-modified FLPA demonstrated the strongest fluorescence, indicating its superior capacity for time-dependent cellular accumulation.

This result was further validated by flow cytometry analysis (Figure 7C). Following 2 h of incubation, the average fluorescence intensity was FL > FLP > FLPA, consistent with the results obtained using inverted fluorescence microscopy. Later, after 6 h of incubation, the results were reversed. FLPA achieved an intensity more than twice that of FL, representing a 135.06% increase over FL, while FLP showed only a 17% improvement. This indicated that ε-PL and SA enhanced the absorption and uptake of hydrosoluble active ingredients during extended incubation periods. Previous studies by Jara-Quijada et al. [51] have shown that chitosan-coated liposomes only modestly improved the bioaccessibility of green tea polyphenols from 31.50% to 35.69%. This limited enhancement may be associated with the large particle size and insufficient protection against gastrointestinal degradation. In contrast, the ELPA system achieved encouraging enhancement in fluorescence intensity compared to the unmodified system.

While the modified polysaccharide slowed down the initial interaction with cells, colloidal stability and residence time in the pericellular space were considerably enhanced. Consequently, more intact vesicles were internalized during prolonged incubation [79]. As cellular uptake is a critical determinant of in vitro bioavailability, the above studies conclusively demonstrated the enhanced potential of bioavailability of EGCG.

4. Conclusions

Herein, a novel EtOH-CAG method was successfully exploited for the active loading of EGCG nanoliposomes with high encapsulation efficiency, small particle size, and PDI in an attempt to overcome the disadvantages of EGCG, such as chemical instability and poor bioavailability, in a green manner. Moreover, the surface of EL was further modified based on the polyelectrolyte properties of ε-PL and sodium alginate. Among these formulations, ELPA represented improved physical stability and in vitro bioavailability compared to EL. The innovative remote loading approach for EGCG nanoliposome established in this work would also be applicable for other bioactive hydrophilic polyphenols as ingredients of functional foods or nutritional supplements.

Nevertheless, the present in vitro assays on evaluation of bioavailability were restricted to cellular uptake experiments in BRL-3A cells, which, while indicative of membrane permeability, did not fully recapitulate the complex physiological conditions of gastrointestinal digestion and systemic absorption. Future research will therefore focus on simulated gastrointestinal digestion and in vivo pharmacokinetic studies to clarify the systemic bioavailability of EGCG delivered by the ELPA system. Furthermore, broadening the applicability of the loading strategy and investigating the interaction of these nanoliposomes within complex food matrices, as well as their long-term storage stability, will be essential to facilitate their industrial application.