Tailored anthocyanin delivery through modulating -COOH groups in pH-responsive peptide gels

Abstract

With the continuous advancement of precision nutrition, researchers have increasingly recognized the need for tailored, stable delivery of food bioactive compounds, such as anthocyanins, in response to the varying conditions of the gastrointestinal (GI) environment. In this proof-of-concept study, we hypothesize that by modulating the number of pH-responsive -COOH groups in peptide gels, we can precisely control their pH responsiveness, rheological properties, and other physicochemical characteristics, enabling efficient anthocyanin delivery under diverse GI conditions. Our experiments demonstrate that increasing the number of -COOH groups and amino acids in the peptide sequence enhances the pH responsiveness, rheological properties, and stability of anthocyanin molecules within the gels, without compromising their biocompatibility or altering their release rate. These results validate our hypothesis and offer a customizable solution for meeting the diverse delivery needs of gastrointestinal environments. It may enrich the tools available for precise nutritional delivery, enhancing their bioavailability and therapeutic potential.

Highlights

• •Stable anthocyanin delivery across diverse applications.
• •Tailored the physicochemical properties of peptide gel carriers.
• •Different peptide gels with differing pH sensitivity for tailored selection.

1. Introduction

As vital bioactive compounds and natural colorants in the food industry, anthocyanins face inherent instability, greatly restricting their broader application. This challenge is particularly evident in their absorption process. Although anthocyanins are primarily absorbed in the small intestine, most ingested molecules are highly susceptible to degradation in the gastric environment, leaving only a minimal amount available for utilization (Ribnicky et al., 2014). Therefore, developing strategies to protect anthocyanins from gastric acid and ensure their stable delivery in the small intestine is essential for enhancing their bioavailability and expanding their potential applications in food and biomedical sciences.

Notably, recent studies have revealed that factors like age, GI diseases, and dietary habits can significantly alter the pH of the GI environment (Zhang, Chen and Campanella, 2024; Zhang et al., 2025). With the advancement of precision nutrition, it has become increasingly clear that a one-size-fits-all approach to anthocyanin delivery is insufficient for effective nutrition delivery (Passarelli et al., 2024). Thus, researchers are now recognizing the need for tailored solutions that address the unique GI environments of different populations (Guo et al., 2017). For example, Abuhelwa et al. (2017) analyzed GI pH levels in 1917 adult samples. They found that factors such as age, GI diseases, and dietary habits significantly influence the pH of the GI tract. In children, the gut microbiota plays a critical role in GI growth and immune system development, with pH changes often accompanying this process (Saeed et al., 2022). Additionally, in individuals with irritable bowel syndrome (IBS), impaired bicarbonate secretion from the pancreas leads to a more acidic pH in the duodenum (pH < 5.5) (Camilleri, 2021). In patients with other GI disorders undergoing pharmacological treatment, the impact of medications on GI pH is a significant concern. For instance, proton pump inhibitors (PPIs) and histamine H2 receptor antagonists, which inhibit gastric acid secretion, can significantly elevate gastric pH (pH > 4) and maintain this elevated pH for at least 12 h or longer (Abuhelwa et al., 2017).

Thus, research on gastrointestinal environments across different populations has demonstrated that pH variation is the key characteristic distinguishing these conditions (Liang et al., 2025). Although several gel materials have been successfully developed for the stable delivery of ANCs under a single pH condition (such as., the peptide-blended whey protein gel by Ozel et al. (2020), and sodium alginate nanogels by Xu et al. (2024)), these systems may be inadequate for abnormal gastrointestinal pH conditions (e.g., in patients with intestinal stress syndrome or the elderly). Therefore, the absence of delivery materials capable of addressing varied gastrointestinal pH environments has emerged as a prominent research gap.

Building on our earlier investigations, we harnessed the natural pH-sensitive charge variations of the -COOH groups found on the side chains of amino acids E and D. In the acidic milieu of the stomach, these groups remain largely neutral, whereas, in the mildly alkaline conditions of the intestine, they ionize to form negatively charged carboxylate ions. This ionization markedly enhances the electrostatic repulsion among the peptide molecules, which disrupts the initially well-organized gel network and promotes the effective release of encapsulated anthocyanins. Consequently, the -COOH groups on the E/D side chains emerge as the molecular cornerstone for the gel's pH responsiveness (Li et al., 2024).

Motivated by this observation, we explored whether modulating the number of -COOH groups in the peptide structure could yield gels with diverse pH-responsive behaviors and tailored physicochemical properties. By varying the number of E residues, our goal was to fine-tune the gel's release properties across different gastrointestinal pH environments, thereby offering a promising solution for the stable delivery of anthocyanins in various nutritional contexts (Fig. 1).

Fig. 1.

A schematic representation of this study illustrates the design of tailored anthocyanin delivery by modulating the number of -COOH groups in peptide gels to accommodate various GI conditions.

2. Materials and methods

2.1. Materials

Morpholine, dimethyl sulfoxide (DMSO), trifluoroacetic acid (TFA), triisopropylsilane (TIS), glucono-δ-lactone (GDL), N, N-diisopropylethylamine (DIPEA), and phosphate-buffered saline (PBS) were all procured from Energy Chemical (Sichuan, China). Hydrochloric acid, methanol, potassium chloride, N, N-dimethylformamide (DMF), chlorine dioxide, diethyl ether, sodium acetate, and sodium carbonate were purchased from Chengdu Cologne Chemical Co., Ltd. (Sichuan, China). Amide-MBHA resin (loading capacity 0.68 mmoL/g) and Fmoc-protected amino acids were acquired from Nanjing Peptide Biotech Co., Ltd. (Jiangsu, China). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was procured from Energy Company (Anhui, China). Anthocyanin (from mulberry, 25 % purity) was obtained from Shengqing Biotechnology Co., Ltd. (Xi'an, China). Simulated gastric fluid (pH 1.5) and simulated intestinal fluid (pH 6.8) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). DMEM, fetal bovine serum (FBS), penicillin, streptomycin, and culture dishes were obtained from Thermo Fisher Scientific (Massachusetts, USA). Fresh, healthy mouse blood cells were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). HEK-293 T cells were sourced from Wuhan Pricella Biotechnology Co., Ltd. (Hubei, China) and cultured in DMEM supplemented with 10 % (v/v) FBS, 100 μg/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in an incubator at 37 °C with 5 % CO₂.

The main instruments and equipment used in this study were HPLC (LC-20, Shimadzu), LCMS (Agilent LCMS 1260–6120, Agilent), Zetasizer (Nano ZS, Malvern), Scanning electron microscopy (Apreo2C, Thermo Scientific, America), Rheology analysis (MCR 302, Anton Paar, China), Fluorescence microscopy image (Ti—S, Nikon, Japan).

2.2. Peptide synthesis

Peptides Fmoc-FE-COOH (F-FE2), Fmoc-FEFE-COOH (F-FE4), Fmoc-FEFEFE-COOH (F-FE6), Fmoc-FEFEFEFE (F-FE8), and Fmoc-FDFDFD-COOH (F-FD6) were synthesized using the standard Fmoc-based solid-phase peptide synthesis (SPPS) strategy. Then, the MBHA resins were mixed with cleavage buffer (trifluoroacetic acid (TFA): ultrapure water (Up): TIS = 9.5:2.5:2.5) for 1.5 h. After filtration, the resin was dried under N₂, and the peptides were precipitated with cold diethyl ether, followed by evaporation. The crude peptides were purified using HPLC, and their molecular weights were confirmed by LC-MS (refer to supplementary material). The purified peptides were lyophilized and stored at −20 °C.

2.3. Preparation of peptide hydrogels

5 mg of each peptide was weighed and mixed with 200 μL of ultrapure water to maintain the ratio of peptide-to-water at 1:40. Sodium carbonate (10 μL) was added, and the mixture was sonicated for 5 min to ensure complete dissolution. Subsequently, GDL (10 mg) was incorporated, and the solution was vortexed and allowed to stand, forming pH-responsive peptide hydrogels.

2.4. Characterization of peptide hydrogels with varying numbers of carboxyl groups

2.4.1. Zeta potential

Each of the five peptides (5 mg) was dissolved in 1 mL of ultrapure (Up) water containing 10 μM sodium carbonate, followed by 5 min of sonication to ensure complete dissolution. Ultrapure water with 10 μM sodium carbonate was used as the blank control. The surface zeta potential was measured at room temperature (25 °C) using a Zetasizer Nano ZS (Malvern).

2.4.2. Surface microscopic morphology

Scanning electron microscopy was used to analyze the microscopic morphology of the peptide hydrogels. Following freeze-drying of the various peptide hydrogels, the samples were fixed using conductive adhesive coated with gold nanoparticles. Finally, the surface microscopic morphology was observed using a scanning electron microscope (Apreo2C, Thermo Scientific, USA) under an accelerating voltage of 5 kV.

2.4.3. Rheology analysis

Peptide hydrogels were prepared according to the method described in Section 2.3. Subsequently, the rheological properties, including mechanical strength and stability, were assessed using a rheometer (MCR 302, Anton Paar, China). Each hydrogel was placed on the rheometer platform, and amplitude sweeps were conducted under controlled conditions using a 25 mm probe. The oscillation frequency was set to 1 Hz, with a strain range of 0.01 % to 100 %. This allowed for the determination of each peptide sequence's storage modulus (G'), loss modulus (G"), and phase angle. Additionally, the mechanical stability of the pH-responsive peptide hydrogels was evaluated by recording changes in G' and G" at a strain of 0.1 % and an angular frequency ranging from 0.1 to 100 rad/s.

2.5. Biocompatibility evaluation

2.5.1. Cell cytotoxicity analysis

Based on previously established research protocols, we evaluated the cytotoxic effects of peptide hydrogels using the MTT method (Cai et al., 2019). HEK-293 T cells were cultured overnight at 1 × 104 cells per well in a 96-well plate under optimal conditions. The hydrogels were then immersed in 1 mL of PBS at 37 °C for 4 h to produce hydrogel eluates. These eluates were added to the cultured cells, while DMEM medium without hydrogel eluate served as the negative control. After an incubation period of 24 h, around 70 μL of fresh medium containing MTT solution was introduced, and the cells were incubated for an additional 3 h at 37 °C. After removing the culture medium, 150 μL of DMSO was introduced to dissolve the precipitated sediment. Absorbance at 570 nm was determined using a microplate reader (SpectraMax 13×, Molecular Devices, USA) to assess the influence of peptide hydrogels on HEK-293 T cell viability.

$$\text{Cellular viability}=\frac{A_{\mathit{sample}}}{A_{\mathit{negative}}}\times \left(100\%\right)$$

2.5.2. Hemolysis toxicity evaluation

The hemolytic toxicity of the peptide hydrogel was assessed using fresh mice erythrocytes, as described in previous studies (Li et al., 2019). The hydrogel samples were incubated in 1 mL of PBS (1×, pH 7.4) at 37 °C for 4 and 8 h, respectively. The eluates were collected and passed through a 0.22 μm filter for further analysis. Fresh erythrocytes were washed and diluted in PBS. Then, the hydrogel eluates (500 μL) were incubated with erythrocytes (5 × 10⁷ cells/mL) at 37 °C for 1.5 h. PBS was employed as the negative control, and 0.1 % sodium dodecyl sulfate (SDS) was used as the positive control. Following incubation, the erythrocytes were centrifuged at 800 rpm for 10 min, and the supernatant was collected. The absorbance of the supernatant was measured at 540 nm using a microplate reader (SpectraMax 13×, Molecular Devices, USA), and the hemolytic activity of the pH-responsive peptide hydrogel was quantified accordingly.

$$\text{Hemolytic ratio}\left(\%\right)=\frac{A_{\mathit{sample}}-A_{\mathit{nagetive}}}{A_{\mathit{positive}}-A_{\mathit{nagetive}}}\times 100\%$$

where A_sample represents the absorbance of peptide hydrogel, A_negative represents the absorbance of negative control (PBS), and A_positive represents the absorbance of positive control (0.1 % SDS).

2.6. Investigation of the loading efficiency of peptide hydrogels

2.6.1. Determination of the anthocyanin content

Following established protocols, the pH differential method determined the anthocyanin concentration (Li, Linli, Yang and Chen, 2023). The anthocyanin solution was diluted in potassium chloride buffer (0.025 M, pH 1.0) and sodium acetate buffer (0.4 M, pH 4.5). Then, the absorbance readings were taken at 520 nm and 700 nm using a microplate reader (SpectraMax 13×, Molecular Devices, USA). The anthocyanin content was calculated using the corresponding formula:

$${\mathrm{C}}_{\left(\mathrm{mg}/\mathrm{L}\right)}=\frac{\left[(A_{520}-A_{700}\right)]{\mathit{pH}}_{1.0}-\left[(A_{520}-A_{700}\right)]{\mathit{pH}}_{4.5}\times M_w\times \mathit{DF}\times 1000\times V}{\epsilon \times L.}$$

2.6.2. Assessment of encapsulation efficiency

In the present study, naturally sourced anthocyanins derived from mulberries were utilized. Building on the techniques reported by He et al. (2017) and Wang et al. (2017). we adopted the pH differential method to determine the anthocyanin content. This method effectively reduces interference from non-anthocyanin molecules present in the extract, thereby facilitating a more precise evaluation of the encapsulation efficiency in the peptide hydrogels. More specifically, 1 mg of anthocyanins derived from mulberries was used to prepare various peptide-anthocyanin hydrogels following the procedures outlined in Section 2.3. After thorough mixing, the supernatant was collected using a 0.45 μm filter membrane. The concentration of unencapsulated anthocyanins in the supernatant was quantified according to the method described in Section 2.6.1. Finally, the encapsulation efficiency of the peptide hydrogel for anthocyanins was calculated using the formula below:

$$\text{Encapsulation Efficiency}\left(\%\right)=\frac{\text{Total anthocyanins}-\text{Surface anthocyanins}}{\text{Total anthocyanin}}\times 100\%$$

2.6.3. Fluorescence microscopy imaging

As natural pigment molecules, anthocyanins exhibit red solid fluorescence under an inverted fluorescence microscope. This characteristic allows for observing the distribution of natural anthocyanin molecules within gel materials using this microscopy technique. In this study, we prepared peptide hydrogels and incorporated mulberry anthocyanins, as described in Section 2.3. After freeze-drying at −80 °C, the samples were analyzed using a fluorescence microscopy system (Ti—S, Nikon, Japan) to examine the microstructural morphology of the hydrogels and assess the morphological changes before and after anthocyanin loading.

2.7. Investigation of anthocyanin stability enhancement by peptide hydrogels

2.7.1. Temperature stability

Based on the analytical evaluation methods established in prior literature (Voss et al., 2023). The peptide-anthocyanin hydrogels were incubated in a water bath at 80 °Cfor 5 h, with a free anthocyanin solution as a control. Then, the remaining anthocyanin content was quantified using the method outlined in Section 2.6.1, and the stability of the anthocyanins in the peptide hydrogels was calculated using the following formula:

$$\text{Retention rate}\left(\%\right)=\frac{{\mathrm{C}}_{\text{Final anthocyanins}}}{{\mathrm{C}}_{\text{Initial anthocyanins}}}\times 100$$

2.7.2. Cu²⁺ stability

Based on the established analytical assessment methods (Yao et al., 2021), the peptide-anthocyanin hydrogels were incubated in different concentrations of CuCl₂ solutions (10 mM and 50 mM) for 3 h, with a free anthocyanin solution serving as the control. Subsequently, the residual anthocyanin content was quantified using the method outlined in Section 2.6.1. The anthocyanin retention rate (%) was calculated according to the formula in Section 2.7.1 to evaluate the efficacy of peptide hydrogels with varying carboxyl group quantities in enhancing anthocyanin stability against metal ions.

2.7.3. Storage stability

Following the analytical assessment method reported by Schüller et al. (2015), equal amounts of peptide-anthocyanin hydrogels and free anthocyanin solutions were stored at room temperature for 30 days. Subsequently, the anthocyanin content remaining in both formulations was assessed using the procedure detailed in Section 2.6.1. The retention rate was calculated using the formula in Section 2.7.1 to analyze the impact of peptide hydrogels on the storage stability of anthocyanins.

2.7.4. Antioxidant activity stability

The impact of peptide-anthocyanin hydrogels on the antioxidant activity of natural anthocyanins was examined following the method described by Liang et al. (2022). Peptide-anthocyanin hydrogels were incubated at 50 °C for 72 h, with a free anthocyanin solution as the control. Subsequently, 50 μL of each sample supernatant was added to 200 μL of ethanol solution containing DPPH (0.4 mM) and allowed to react in the dark at room temperature for 30 min. Then, the absorbance at 517 nm was measured, and the DPPH radical scavenging efficiency for each sample was calculated using the following formula to assess the influence of different peptide hydrogels on anthocyanin antioxidant activity.

$$\text{DPPH reduction}\left(\%\right)=\left(1-\frac{A_1-A_2}{A_0}\right)\times 100\%$$

Here, A0 indicates the absorbance at 517 nm for the blank, composed of 200 μL of DPPH ethanol solution and 50 μL of ethanol. A1 is the absorbance at 517 nm for the sample containing 200 μL of DPPH ethanol solution and 50 μL of anthocyanin solution. A2 denotes the absorbance at 517 nm for the sample without DPPH, consisting of 200 μL of ethanol and 50 μL of anthocyanin solution.

2.8. Controlled anthocyanin release in stimulated gastric (intestinal) fluid environment

In accordance with the experimental approach outlined by Yuan et al. (2024), we examined the release dynamics of anthocyanin-loaded peptide hydrogels in simulated gastrointestinal conditions. The experimental procedure was conducted as follows: Peptide-anthocyanin hydrogel samples, each containing 1 mg of natural anthocyanins, were immersed in 1 mL of simulated gastric fluid (SGF: 0.2 % NaCl, 3.2 mg/mL pepsin, pH = 1.5 ± 0.1) and simulated intestinal fluid (SIF: 0.5 mg/mL trypsin, 0.7 mg/mL bile extract, 1.7 % Na₂HPO₄, pH = 6.8 ± 0.1). The samples were thoroughly mixed and incubated at 37 °C for 2 h in SGF and 10 min in SIF to simulate physiological conditions. Following incubation, the samples were centrifuged at 5000 rpm for 5 min, and the supernatants were collected for anthocyanin release analysis using the pH differential method, as detailed in Section 2.6.1. The anthocyanin concentration in the supernatant was determined and used to calculate the release rate of ANCs from the hydrogel using the following formula:

$$\text{Release rate}\left(\%\right)=\frac{{\mathrm{M}}_{\text{Final anthocyanins}}}{{\mathrm{M}}_{\text{Initial anthocyanins}}}\times 100\%$$

A key aspect of our analysis was capturing the rapid pH response of the hydrogel in intestinal fluid. Given its high sensitivity to pH changes, we used a stopwatch to precisely record the time required for the hydrogel to dissolve completely, transitioning from a gel to a solution state.

2.9. Statistical analysis

All experiments were conducted in triplicate, and results are reported as mean ± standard deviation. Data analysis was performed using one-way analysis of variance (ANOVA). Figures were processed with Origin 2018 (OriginLab Co. Ltd., USA), Chemdraw (CambridgeSoft, USA), and Microsoft PowerPoint (Microsoft, USA). Statistical significance is indicated as ns (not significant) and ***p < 0.001.

3. Results and discussion

The pH-responsive behavior originates from glutamic acid (E) or aspartic acid (D) residues in the peptide structure. These amino acids contain carboxyl (-COOH) groups that undergo protonation-deprotonation transitions corresponding to environmental pH. In acidic gastric conditions (<pH 3), protonated carboxyl groups maintain electrical neutrality, facilitating parallel peptide alignment and formation of a three-dimensional hydrogel network for anthocyanin encapsulation. Conversely, under intestinal alkaline conditions (pH 7–8), deprotonation generates carboxylate anions (-COO⁻), creating electrostatic repulsion between adjacent peptide chains. This repulsion disrupts molecular packing and triggers gel matrix disintegration, enabling controlled anthocyanin release (Panja et al., 2021).

Therefore, building upon fundamental principles of peptide self-assembly (Panja et al., 2021), our hydrogel design adheres to three biochemical principles: (1) pH responsiveness through glutamic/aspartic acid (E/D) selection, as these uniquely possess ionizable -COOH groups among proteinogenic amino acids. (2) Hydrophobic stabilization via aromatic residues (F/W), excluding histidine due to its hydrophilic imidazole moiety. (3) Optimized molecular packing using phenylalanine (F) for its optimal van der Waals radius and π-π stacking potential (Tena-Solsona et al., 2014). Moreover, to counterbalance hydrophilic/hydrophobic equilibrium, each E residue was systematically paired with F. This strategy generated four derivatives: F-FE2, F-FE4, F-FE6, and F-FE8. Additionally, we designed F-FD6 as an isofunctional analog containing equivalent -COOH groups but differing in backbone composition.

3.1. Investigation of physicochemical properties

The structure-property relationship is a fundamental principle governing molecular behavior. For peptide molecules with varying numbers of -COOH groups, their physicochemical properties play a critical role in the loading and stabilization of anthocyanin molecules. Therefore, we first investigated the prepared peptide gels from four critical perspectives: pH responsiveness, electrostatic potential, microstructure, and rheological properties.

3.1.1. pH responsiveness

We first examined the pH-responsive behavior of the gels using HCl and NaOH solutions. As shown in Fig. 2 and Fig. S1, under the pH conditions corresponding to F-FE2, F-FE4, F-FE6, and F-FE8 the acid-base indicator bromocresol green (pH range 3.6–5.4) exhibited a gradual color transition from grass green to blue. Impressively, peptides with varying numbers of -COOH groups and amino acids demonstrated distinct pH responses. Notably, as the number of -COOH groups and amino acids increased, the gels' pH response shifted toward more alkaline conditions as the number of -COOH groups increased. We hypothesize that the shift in pH responsiveness is caused by stronger intermolecular interactions (such as hydrogen bonding between peptide backbones) as the number of -COOH groups increases. This may result in the need for a higher concentration of negatively charged carboxylate ions (-COO⁻) to disrupt the orderly arrangement of the peptide molecules, facilitating the transition from a gel to a liquid state. Furthermore, controlled experiments with structurally identical peptides containing different side chain groups (Fmoc-FDFD-NH₂ [-COOH] versus Fmoc-FSFS-NH₂/Fmoc-FLFL-NH₂ [-OH or isopropyl]) confirm that the pH-responsive behavior of peptide hydrogels originates specifically from carboxyl group variations rather than amino acid residues (Fig. S2-S3). It is noteworthy that once the critical pH is surpassed, the transition from gel to liquid occurs within 23 s, with negligible differences in response time (refer to supplementary material). These findings indicate that the pH response is highly sensitive for the FE series peptide gels, with even slight changes in surface charge (such as, F-FE2) triggering a rapid transition.

Fig. 2.

The morphological images of pH-responsive peptide hydrogels at their critical pH (bromocresol green pH indicator: yellow color signifies pH < 3.8, while blue color represents pH > 5.4). (A) and (E) illustrate the behavior of F-FE2 near the critical pH transition threshold (lipid and gel). (B) and (F) illustrate the behavior of F-FE4 near the critical pH transition threshold (lipid and gel). (C) and (G) depict the response of F-FE6 near the critical pH transition threshold (lipid and gel). (D) and (H) depict the response of F-FE8 near the critical pH transition threshold (lipid and gel). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

We further measured the precise pH responsiveness of the peptide gels using a pH meter. Excitingly, these peptide molecules followed a general trend: the greater the number of -COOH groups, the higher the corresponding pH value. Specifically, the pH values for F-FE2, F-FE4, F-FE6, and F-FE8 were 4.5, 5.3, 6.0, and 7.2 respectively. Additionally, the distribution of -COOH groups within the peptide structure (i.e., the type of amino acids) also impacted the gels' pH responsiveness. For instance, despite having similar structures, peptides containing D-amino acids demonstrated a more acidic pH response than those with E-amino acids (F-FE6 6.0, F-FD6 5.1). This difference may be attributed to variations in the intermolecular interactions between F-FD6 and F-FE6. Overall, these findings suggest that the pH responsiveness of hydrogels can be precisely controlled by adjusting both the number of -COOH groups and their molecular environment within the peptide structure.

3.1.2. Zeta potential

In the design of peptide gels, the negatively charged carboxylate ions (-COO⁻) formed by the side-chain -COOH groups of Glu (E) and Asp (D) in alkaline environments play a key role in disrupting the ordered arrangement of peptide molecules, which ultimately causes to the collapse of the gel structure. Thus, we performed Zeta potential analysis to further investigate the surface properties of these pH-responsive peptide gels under mildly alkaline conditions. As shown in Fig. S4, a pattern similar to the pH responsiveness was observed: for structurally similar peptide molecules, increasing the number of -COOH groups resulted in a gradual decrease in surface Zeta potential. Specifically, F-FE8, which contains the highest number of -COOH groups, exhibited a Zeta potential as low as −50 mV. Notably, although the increase of -COOH groups significantly reduced the surface potential, which could theoretically affect the gel's response rate, the transition from gel to liquid state is also strongly influenced by the intermolecular interactions between peptide molecules. This ultimately raises the pH response threshold for the peptide hydrogels.

3.1.3. Microstructure characterization

Unlocking the microscopic architecture of peptide gels through SEM reveals a promising foundation for stable anthocyanin delivery. Fig. 3 shows a cleat three-dimensional network across different types of peptide gels, echoing the organized structures found in hydrogels. This consistency confirms the successful formation of peptide gels and points to possibilities for controlled nutrient delivery applications.

Fig. 3.

Micromorphology characterization of peptide gels with diverse pH responsive properties.

3.1.4. Rheology analysis

To serve as an effective delivery material for stabilizing anthocyanins, the gels must exhibit sufficient mechanical strength to protect the anthocyanin molecules from various environmental stresses. Therefore, rheological properties are a crucial consideration in the design of anthocyanin-stabilizing gels. In this study, we further analyzed the differences in the rheological behavior of peptide gels containing varying numbers of -COOH groups. As shown in Fig. 4 and Fig. S5, none of the storage modulus (G') and loss modulus (G") curves for the peptide hydrogels intersected, and all tan δ values were less than 1, indicating that the loss modulus was lower than the storage modulus. These results suggest that all peptide hydrogels formed a three-dimensional network structure with some rheological elasticity (Struck et al., 2018). Notably, as the number of -COOH groups and amino acids increased, the storage modulus (G') and loss modulus (G") of the gels significantly improved (F-FE2 < F-FE4 < F-FE6 < F-FE8). Additionally, the tan δ values for F-FE2, F-FE4, F-FE6, and F-FE8 showed a gradual decline, indicating a transition toward a more viscous behavior and an increase in gel strength (Bauland et al., 2022). This may be attributed to the progressively stronger hydrogen bonding interactions between peptide backbones as the number of -COOH groups and amino acids increases. For example, the hydrogen bonds between F-FE2 molecules (4H-bonds) increase to 16H-bonds in F-FE8 molecules, leading to enhanced mechanical properties of the peptide gels on a macroscopic scale. This observation aligns with the trend that peptides with more -COOH groups require higher pH environments to transition from a gel to a liquid state. Additionally, the specific amino acid composition could influence the rheological properties of peptide gels, as seen with F-FE6 and F-FD6. This may be attributed to changes in the side-chain structure of the amino acids, which alter the alignment of the peptide gel molecules. These findings suggest that by modulating the number of -COOH groups or amino acids, the mechanical properties of pH-responsive peptide gels can be fine-tuned without affecting their pH sensitivity. This approach enables customization of the gel's mechanical properties to meet specific application requirements.

Fig. 4.

Rheological investigation of peptide gels with varying numbers of -COOH groups (amplitude sweep analysis). (A) Fmoc-FE-COOH; (B) Fmoc-FEFE-COOH; (C) Fmoc-FEFEFE-COOH; (D) Fmoc-FEFEFEFE-COOH.

3.2. Investigation of biocompatibility

High biocompatibility is essential for ensuring the stable and safe delivery of anthocyanin molecules. As endogenous biological molecules, peptide-based materials have proven to be well-suited for the stable delivery of various functional nutritional factors in food systems. In this study, we further assessed the biocompatibility of F-FE2, F-FE4, F-FE6, and F-FD6 by evaluating their cytotoxicity and hemolytic toxicity.

3.2.1. Cytotoxicity

To assess cytotoxicity, the MTT assay was employed to determine the impact of peptide gels on the growth of HEK-293 T cells. As illustrated in Fig. 5A, compared to the negative control (PBS), the viability of HEK-293 T cells exposed to the peptide gel extracts remained consistently above 90 %, with no significant reduction in cell viability. These findings indicate that the peptide-based gel materials do not negatively affect cell growth and exhibit negligible cytotoxicity (Xiang et al., 2022).

Fig. 5.

Biocompatibility analysis of peptide gels with varying numbers of -COOH groups. (A) Cytotoxicity analysis with HEK-293 T cells; (B) Hemolysis analysis with blood cells. The results were analyzed by one-way analysis of variance (ANOVA), and the level of significance were indicated as: ns (no significance), and ***p < 0.001.

3.2.2. Hemolytic toxicity

The hemolysis assay is an effective method for assessing the compatibility of biomaterials with blood cells by measuring the degree of red blood cell lysis and hemoglobin release. Generally, a hemolysis rate below 5 % is considered indicative of low hemolytic toxicity (Xiang et al., 2022). As shown in Fig. 5B, after 1.5 h of incubation at 37 °C, none of the peptide gel test groups exhibited a significant increase in absorbance at 540 nm compared to the positive control (0.1 % SDS), indicating no damage to the red blood cells. These findings demonstrate that none of the peptide gels exhibit noticeable hemolytic toxicity.

In conclusion, altering the number of -COOH groups and amino acids in peptide structure does not compromise their biocompatibility. These developed peptide gels could effectively fulfill the safety requirements for the stable delivery of anthocyanins and other nutritional molecules in food applications.

3.3. Effective encapsulation of anthocyanins

3.3.1. Encapsulation efficiency

To achieve effective and stable delivery of anthocyanin molecules, gel materials must first encapsulate them within their three-dimensional network structure. Therefore, we investigated the loading capacity of various peptide gels for anthocyanins using pH differential methods. Notably, all peptide hydrogels demonstrated a satisfactory encapsulation efficiency exceeding 90 %. Specifically, the encapsulation rates for F-FE2, F-FE4, F-FE6, and F-FD6 were 97.8 % ± 1.0, 97.8 % ± 0.1, 97.4 % ± 0.1, and 93.3 % ± 0.3, respectively. These results indicate that the peptide gel materials possess a high loading capacity for anthocyanin molecules, suggesting their potential as stable delivery systems.

3.3.2. Anthocyanins distribution

As natural fluorescent molecules, anthocyanins allow easy observation of their distribution within peptide gels using inverted fluorescence microscopy (Zhang et al., 2015). In Fig. 6, the bright-field image reveals the glassy fibrous structure of the peptide gel, verifying successful gel formation. When anthocyanins were incorporated into the peptide gels, a distinct red fluorescence was observed, uniformly distributed throughout the gel matrix under both bright-field and fluorescence modes. Additionally, no fluorescence aggregation was detected, suggesting that the anthocyanin molecules were evenly and effectively encapsulated within the peptide gels.

Fig. 6.

Inverted fluorescent imaging of encapsulated anthocyanin in peptide gels (fluorescent mode and bright-field). (A-B) F-FE2; (C—D) F-FE4; (E-F) F-FE6.

3.4. Evaluation of anthocyanin stability improved by peptide gels

Anthocyanin molecules are highly prone to inactivation and degradation when exposed to environmental stressors (such as elevated temperatures and metal ions). This sensitivity is a significant limitation to their applications in food and biomedical fields, underscoring the need for stable delivery solutions. Consequently, we further evaluated the protective effects of various peptide gels on anthocyanins, assessing their resilience against environmental stresses, specifically in terms of high temperature, metal ion exposure, storage stability, and antioxidant activity.

3.4.1. Thermal stability

As shown in Fig. 7A, after incubation at 80 °C for 5 h, anthocyanins stabilized within peptide gels exhibited significantly higher retention rates than those in the free anthocyanin solution. Specifically, the F-FE6 gel demonstrated a retention rate exceeding 70 %. Notably, the stabilization effect of the peptide gels on anthocyanins under high-temperature conditions increased with the number of -COOH groups and amino acids present. For instance, the retention rates of anthocyanins in F-FE2, F-FE4, and F-FE6 gels were 54.2 % ± 0.2, 68.0 % ± 0.3, and 70.9 % ± 0.2, respectively, consistent with the mechanical trends observed in rheological analyses. This improved retention is likely due to the enhanced mechanical strength of the peptide gels, which provides better protection for encapsulated anthocyanins against high-temperature stress. Additionally, as illustrated in Fig. S6, no significant morphological changes were observed in the peptide gels, regardless of the presence of ANC molecules, before or after high-temperature incubation. The gels maintained their structural integrity, suggesting that the peptide gels exhibit stability under thermal stress, thereby effectively safeguarding the encapsulated ANC molecules (Mao et al., 2023).

Fig. 7.

Investigation of anthocyanin stability improved by peptide gels. (A) Thermal stability investigation under 80 °C for 5 h. (B) Metal ion stability evaluation under 10 mM and 50 mM Cu²⁺ stress. (C) Long-term storage stability for 34 days with or without light. (D) Effective protection on anthocyanin antioxidant activity (DPPH) at 50 °C. The results were analyzed by one-way analysis of variance (ANOVA), and the level of significance were indicated as: ns (no significance), and ***p < 0.001.

3.4.2. Cu²⁺ stability

In practical applications, anthocyanin molecules are prone to inactivation under exposure to heavy metal ions, such as Cu²⁺. To address this challenge, we further investigated the stability of anthocyanins encapsulated in peptide gels under Cu²⁺ stress at varying concentrations. As shown in Fig. 7B, after 3 h of exposure to 10 mM and 50 mM Cu²⁺, a significant loss of unprotected anthocyanins occurred, with retention rates dropping to only 48.5 % ± 0.1 and 26.5 % ± 0.3, respectively. In contrast, anthocyanins encapsulated within peptide gels maintained much higher retention rates, exceeding 70 % and 40 % under the same conditions.

Notably, under both 10 mM and 50 mM Cu²⁺ concentrations, anthocyanin retention rates improved progressively with peptide gels of increasing -COOH groups, specifically F-FE2, F-FE4, and F-FE6. The retention rates observed were 82.0 % ± 0.14, 94.5 % ± 0.09, and 96.9 % ± 0.03 at 10 mM Cu²⁺, and 51.6 % ± 0.13, 59.7 % ± 0.11, and 59.95 % ± 0.67 at 50 mM Cu²⁺, respectively. These findings indicate that the protective capability of peptide gels on anthocyanins under Cu²⁺ stress increases with the number of -COOH groups and amino acids, consistent with our observations of their rheological properties and thermal stability. Additionally, the type of amino acid also affects the stability conferred by the peptide gel, as seen with F-FE6 and F-FD6. As shown in Fig. S7, the peptide gels maintained their structural integrity after incubation with various concentrations of Cu²⁺, regardless of whether they contained ANC molecules, with no significant morphological changes. These findings indicate that the peptide gels exhibit inherent stability under Cu²⁺ stress, which enables them to effectively protect the encapsulated ANC molecules (Mao et al., 2023).

3.4.3. Storage stability

We further assessed the stability of anthocyanin molecules encapsulated within various peptide gels during long-term storage. As illustrated in Fig. 7C, after one month (34 days) of storage at room temperature, the retention rate of unprotected anthocyanins dropped significantly, reaching only 25.2 % ± 0.1 in light-protected conditions and 25.2 % ± 0.6 without light protection. In contrast, anthocyanins stabilized within peptide gels showed significantly improved retention, with rates ranging from 46.5 % ± 0.6 to 72.3 % ± 1.3. These results indicate that peptide gels with varying numbers of -COOH groups and amino acids are adequate for the prolonged storage of anthocyanins.

3.4.4. Antioxidant activity

Antioxidant activity is a critical biological property of anthocyanins as functional food factors, removing reactive oxygen species (ROS) from the body, maintaining cellular redox balance, and slowing aging (Zhu et al., 2022). Consequently, we further investigated the protective effect of peptide gels on the antioxidant activity of anthocyanins under heat stress at 50 °C using the DPPH method. As shown in Fig. 7D, the anthocyanins stabilized within peptide gels displayed significantly higher antioxidant activity than unprotected anthocyanin solutions.

3.5. Responsive release in gastrointestinal fluid

The instability of anthocyanin molecules in gastric fluid is a key factor contributing to their low bioavailability. Therefore, achieving a stable, safe, and controllable delivery system requires peptide gels that could both protect anthocyanins from degradation in the gastric environment and enable effective release in the intestinal environment. Accordingly, we further examined the release behavior of peptide gels with varying numbers of -COOH groups and amino acids in simulated gastric and intestinal fluids.

3.5.1. Stable behavior in gastric fluid

As shown in Fig. 8A, after 2 h of incubation in a simulated gastric fluid, peptide gels with an FE structure demonstrated significant stabilizing effects on anthocyanins, with the F-FE6 exhibiting a release rate as low as 9.2 % ± 0.4. Impressively, as the number of -COOH groups and amino acids increased, the stabilizing effect of the peptide gels on anthocyanin molecules in gastric fluid progressively improved. Precisely, the anthocyanin release rates followed the trend: F-FE2 (14.7 % ± 0.4) > F-FE4 (11.8 % ± 0.4) > F-FE6 (9.2 % ± 0.4), consistent with the rheological and stability profiles previously observed. We attribute these differences in stabilization to variations in the initial -COOH group and amino acid content impart distinct physicochemical properties to each gel, ultimately determining their protective effects on anthocyanins under gastric conditions.

Fig. 8.

Effective stabilization and responsive release of peptide gels in GI environment. (A) Effective stabilization of anthocyanin in stimulated gastric fluid (SGF) for 2 h; (B) Responsive release of anthocyanin in simulated intestinal fluid (SIF) at slightly acidic (pH 5.5) environment. (C) Responsive release of anthocyanin in SIF at neutral (pH 6.8) environment.

3.5.2. Responsive release in intestinal fluid

Subsequently, we investigated the release behavior of various peptide gels in simulated intestinal fluids at different pH levels: slightly acidic (pH 5.5) and neutral (pH 6.8). As shown in Fig. 8B, the release behaviors at pH 5.5 varied significantly among the gels, reflecting differences in their pH responsiveness (F-FE2 < F-FE4 < F-FE6). Specifically, gels with greater sensitivity to acidic responsive pH showed higher release rates at pH 5.5. For instance, F-FE2 displayed the highest release rate (94.6 % ± 1.5), followed by F-FE4 (79.1 % ± 1.0) and F-FE6 (67.6 % ± 0.7). This behavior can be attributed to the gel-to-liquid transition of F-FE2 occurring at a lower pH, which facilitates the earlier release of encapsulated anthocyanin molecules in acidic environments.

In contrast, as the number of -COOH groups and amino acids in the peptide structure increases, the intermolecular interactions strengthen, shifting the responsive pH toward more alkaline values. Consequently, F-FE4 and F-FE6 demonstrate lower release rates in acidic conditions due to their reduced sensitivity at lower pH levels. Furthermore, it could be anticipated that as the environmental pH decreases further, the release rate of anthocyanins from the peptide gels would decline correspondingly, as observed under gastric conditions (pH = 1.5; Fig. 8A).

At pH 6.8, which exceeds the peptide gels' responsive pH, the release efficiency exceeded 80 % across all samples. Notably, gels with a higher number of -COOH groups achieved a more complete release of anthocyanins in the intestinal environment: F-FE6 (96.1 % ± 0.5) > F-FE4 (95.2 % ± 0.4) > F-FE2 (84.8 % ± 0.3) (Fig. 8C). We propose that the stronger intermolecular interactions in F-FE6 result in a more organized gel structure, facilitating a more effective gel-to-liquid transition under these conditions. These findings confirm that modifying the -COOH groups in pH-responsive peptide molecules enables controlled physicochemical properties and release behavior in both gastric and intestinal fluids.

Systematic evaluation identified persistent anthocyanin (ANC) retention in peptide hydrogel carriers compared to solution-based systems achieving near-complete payload liberation. We believe this discrepancy may stem from the hydrogel's inherently robust mechanical properties, which seem to impede the complete breakdown of its network (Chen et al., 2025). Specifically, the electrostatic repulsion among peptide molecules does not entirely dismantle the gel structure, resulting in some ANCs remaining encapsulated. Drawing inspiration from the solution-based systems, our future work will focus on developing peptide microgels (Pellá et al., 2020). By reducing gel network mechanical strength through chemical modifications, we aim to enhance pH-triggered structural collapse efficiency, thereby minimizing residual ANC retention under intestinal conditions.

4. Conclusions

In an era where bioactive compounds' stable and efficient delivery is crucial for advancing human health, this proof-of-concept study explores a novel approach to enhance the delivery of anthocyanins in complex gastrointestinal environments. We hypothesize that by fine-tuning the number of -COOH groups in peptide-based gels, we can precisely control their pH-responsive properties and mechanical performance. This strategy opens the door to the design of highly adaptable peptide gel carriers, each with tailored physicochemical properties, capable of delivering anthocyanins effectively under varying digestive conditions. Our findings reveal that increasing the number of -COOH groups and amino acids in the peptide sequence could strengthen the non-covalent interactions between peptide molecules, leading to gels with improved mechanical strength, enhanced stability of anthocyanins, and a shift in the pH at which the release occurs. Remarkably, these adjustments do not compromise the gels' biocompatibility or release rate.

Compared to other peptide-based ANCs delivery systems, our pH-responsive peptide gel offers three innovative advantages. First, our gel carrier offers a rapid response to changes in pH; when the environment reaches the designated pH, the gel structure swiftly collapses and transforms into a peptide solution, thereby enabling the efficient release of the encapsulated ANCs. Second, the carrier exhibits tunable pH responsiveness and rheological properties that can be readily adjusted through peptide modifications, ensuring stable delivery across varying gastrointestinal environments. Third, the gel enhances ANCs stability by protecting them from high-temperature and metal ion stresses. In summary, this proof-of-concept study provides a viable strategy for optimizing gel performance and developing multifunctional platforms for targeted delivery of bioactive compounds, such as anthocyanins.

CRediT authorship contribution statement

Wenjun Li: Writing – review & editing, Writing – original draft, Resources, Project administration, Methodology, Conceptualization. Qianqian Bie: Resources, Methodology, Investigation, Formal analysis, Data curation. Jianling Li: Methodology, Investigation. Wenjie Yang: Investigation, Data curation. Pengliang Cao: Investigation, Data curation. Wenyu Yang: Supervision, Resources, Funding acquisition. Xianggui Chen: Supervision, Methodology. Pengfei Chen: Supervision, Methodology.

Declaration of competing interest

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

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.