A dual absorption pathway of novel oyster-derived peptide-zinc complex enhances zinc bioavailability and restores mitochondrial function

Graphical abstract

Highlights

• •A novel oyster-derived peptide-zinc complex enhances zinc bioavailability.
• •IE-Zn complex utilizes dual absorption pathways, improving zinc retention and uptake.
• •IE-Zn reduces oxidative stress and apoptosis, offering superior protection over ZnSO₄.
• •IE-Zn activates AMPK/PGC1-α/NRF-1/TFAM to improve energy metabolism disorders.
• •IE-Zn promotes mitochondrial biogenesis and ATP production, and restores mitochondrial function.

Abstract

Zinc deficiency is a global health issue that impairs immune function, growth, and energy metabolism. Although conventional zinc supplements have been developed, their effectiveness is limited by poor bioavailability and susceptibility to dietary inhibitors. In this study, a peptide-zinc complex (IE-Zn) derived from oysters was developed to enhance zinc uptake and address metabolic disruptions caused by deficiency. It was determined that Zn²⁺ binds with high affinity to the IE peptide, promoting structural flexibility that facilitates zinc transport through both zinc ion transporters and oligopeptide transporters. In Caco-2 and IEC-6 cell models, IE-Zn was shown to significantly improve zinc absorption and retention compared to ZnSO₄, driven by the upregulation of ZIP4 and PEPT1 transporters. In vivo studies in a zinc-deficient mouse model confirmed enhanced zinc absorption and distribution across serum, intestine, and liver. Moreover, IE-Zn restored energy homeostasis by activating the AMPK/PGC1-α/NRF-1/TFAM signaling pathway, promoting mitochondrial biogenesis and reducing oxidative stress. These findings suggest that IE-Zn is a superior zinc supplement with higher bioavailability and serves as a potent regulator of cellular energy metabolism, offering therapeutic potential for managing conditions related to zinc deficiency and mitochondrial dysfunction. This study lays the foundation for further exploration of peptide-mineral complexes as advanced nutritional supplements with broad applications. Subsequent studies will further investigate the absorption pathway and targeting of peptide-zinc complex. The hope is to provide potential preventive applications for people in need, including zinc deficiency and a range of diseases caused by zinc deficiency.

Introduction

Zinc (Zn) is an indispensable trace element vital to numerous physiological processes, functioning as a cofactor for hundreds of enzymes and contributing to the structural stability of numerous proteins. As the second most abundant transition metal in the human body, zinc is critical for immune system function, DNA synthesis, wound healing, and cellular energy metabolism [1]. Given its multifaceted involvement in health, optimal zinc status is essential for overall physiological well-being. Despite its importance, zinc deficiency remains a significant global public health concern, affecting an estimated 17 % of the world’s population, with higher prevalence in developing countries [2]. The consequences of zinc deficiency are far-reaching, leading to a range of health complications, including growth defects, nervous system damage, and immune system deficiencies [3]. For instance, zinc deficiency can result in thymic atrophy, diminished T-cell proliferation, and altered cytokine production, all of which compromise the immune system's ability to respond effectively to pathogens.

The challenge of inadequate zinc intake is exacerbated by the low bioavailability of dietary zinc. Firstly, grains and aquatic foods which are rich in zinc ions are less consumed in diet [4]. Secondly, zinc absorption can be hindered by dietary inhibitors, particularly phytates found in many plant-based foods, which bind zinc and reduce its bioavailability [5]. As a result, populations reliant on plant-based diets may be particularly vulnerable to zinc deficiency. To address zinc deficiency, conventional supplementation strategies typically involve inorganic salts (e.g., zinc sulfate) and organic compounds (e.g., zinc gluconate). Inorganic salts as the first generation of zinc supplements, including zinc sulfate, zinc oxide, zinc carbonate and so on. Although effective, inorganic zinc salts often irritate the gastrointestinal tract, causing adverse reactions such as nausea and vomiting, and have limited bioavailability. Organic compounds are second-generation zinc supplements, such as zinc gluconate, zinc oxalate, zinc citrate, zinc lactate, etc. Compared with inorganic zinc, organic zinc reduces gastrointestinal irritation and improves utilization. Organic zinc salts offer increased bioavailability, but still face limitations, including their reliance on standard absorption pathways that may be inhibited by other dietary factors, reducing overall effectiveness [6]. For example, phytic acid in the diet has a strong affinity with zinc. During the digestion of food, phytic acid forms phytate with zinc, resulting in decreased zinc absorption. At the same time, dietary calcium, iron and other metal ions can also reduce zinc absorption through competitive binding metal ion transport channels [7]. This situation highlights the urgent need for more effective, safe, bioavailable zinc supplements, as well as other transport pathways to promotes zinc absorption.

Recent advances in nutrition science have introduced peptid-zinc complexes as a promising third-generation supplementation strategy that could have better bioavailability than the previous two generations of zinc supplements by improving the solubility and stability of zinc in the gut [8]. Peptide-zinc complexes leverage the metal-binding capabilities of specific bioactive peptides, which act as carriers to stabilize and transport zinc ions. These complexes, particularly those derived from marine organisms, provide unique advantages over traditional zinc sources [9]. Unlike inorganic or organic zinc salts, peptide-zinc complexes are resistant to precipitation with phytates, ensuring stable absorption even in challenging dietary conditions [5]. There are two main absorption pathways of ions in the intestine, which are transcellular and paracellular [10]. Currently, the main pathways known to transport peptides through the intestinal cells are the PEPT1 Pathway, the cell-penetrating peptide pathway and the paracellular pathway [11]. Furthermore, research indicates that these complexes can be absorbed through specialized transport pathways in the intestinal epithelium, such as oligopeptide transporters, which bypass the conventional zinc ion transporters and allow for more efficient uptake [12]. However, it remains unclear how these peptide-zinc complexes might engage alternative cellular transport pathways beyond the traditional ion channels or transporters and to what extent these dual pathways can enhance zinc bioavailability under zinc-deficient conditions. Addressing this question could offer deeper insights into the mechanisms by which peptide-zinc complexes facilitate more efficient zinc absorption.

Marine organisms, especially oysters (Crassostrea gigas), are renowned for their high zinc content [13] and the presence of peptides with exceptional zinc-binding capacity. Oysters, with zinc concentrations reaching up to 5 ‰ of dry tissue weight, represent a superior natural zinc source compared to most terrestrial foods [14]. At the same time, oyster is one of the dual-use materials approved by the Chinese Ministry of Health. It has been found that oyster peptide-zinc complex not only has good solubility and gastrointestinal stability [15], it also has good antioxidant activity [16] and the activity of inhibiting tumor growth [17]. Preliminary studies by our research group have identified a specific dodecapeptide (IEELEEELEAER, referred to as IE) from a variety of Marine organisms such as coiled coil scallops, blue mussels and oysters. IE has good biological activity, including antithrombotic and osteogenic [18], [19]. It has a high affinity and better capacity for zinc ions over other metal ions [20]. By utilizing zinc ion transport and peptide transport mechanisms, the IE-Zn complex demonstrates a dual absorption pathway that improves zinc bioavailability beyond the capabilities of traditional supplements. Given these unique absorption properties, this study hypothesizes that the IE-Zn complex improves zinc bioavailability and mitochondrial function through a dual absorption pathway involving zinc ion transporters (e.g., ZIP4) and peptide transporters (e.g., PEPT1). Furthermore, IE-Zn is postulated to restore mitochondrial function by activating the AMPK/PGC1-α/NRF-1/TFAM signaling axis, which regulates energy homeostasis, mitochondrial biogenesis, and oxidative stress mitigation. By leveraging these mechanisms, IE-Zn may offer significant advantages over conventional zinc supplements.

The structural and functional properties of the IE-Zn complex were investigated, with a focus on its dual absorption pathways and potential for regulating energy metabolism under zinc-deficient conditions. Using Caco-2 and IEC-6 cell models, as well as an in vivo zinc-deficient mouse model, IE-Zn was observed to significantly enhance zinc uptake via both zinc ion transporters and oligopeptide transporters. This dual absorption mechanism, as confirmed by inhibitor assays, allows IE-Zn to bypass common dietary inhibitors like phytates, resulting in higher zinc bioavailability compared to ZnSO₄. Furthermore, IE-Zn was shown to restore energy homeostasis by activating the AMPK/PGC1-α/NRF-1/TFAM signaling axis, a pathway that regulates cellular energy metabolism and mitochondrial function under stress conditions, which is critical for mitochondrial biogenesis. In IEC-6 cells under zinc-deficient conditions, mitochondrial membrane potential and ATP production were significantly improved by IE-Zn, surpassing the effects of ZnSO₄. These findings highlight IE-Zn's dual benefits: not only does it enhance zinc absorption efficiency, but it also addresses metabolic disruptions induced by zinc deficiency, positioning IE-Zn as a promising alternative in zinc supplementation strategies.

Materials and methods

Chemicals

The IE peptide and oyster peptide were obtained from Shanghai Qiangyao Biotechnology Co., Ltd. (Shanghai, China) and Dezhou Lanli Biotechnology Co., Ltd. (Shandong, China), respectively. Zinc sulfate (ZnSO₄₎ and bovine serum albumin (BSA) were purchased from Aladdin Co., Ltd. (Shanghai, China). High- glucose DMEM and MEM were obtained from Thermo Fisher Technologies (Massachusetts, USA), and fetal bovine serum (FBS) from Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China). NVS-ZP7-4 and cytochalasin-D were purchased from MedChemExpress (MCE, new Jersey, USA). Various assay kits, including BCA protein, alkaline phosphatase (ALP/AKP) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). CCK8 kit, reactive oxygen species (ROS) detection kit Annexin V-FITC apoptosis detection kit, mitochondrial membrane potential test kit, ATP test kit, Radio Immunoprecipitation assay (RIPA), along with phenylmethanesulfonyl fluoride (PMSF) and m-βcd were sourced from Beyotime Biotechnology Co., Ltd. (Jiangsu, China). Additional reagents included the EastepTM RNA Extraction Kit from Promega Biotechnology Co., Ltd. (Beijing, China), and multiple PCR kits from Tiangen Biochemical Technology Co., Ltd. (Beijing, China). The primary antibodies against ZIP4, DMT1, ZNT1, β-ACTIN and Goat Anti-Rabbit IgG H&L antibody were purchased from Bioss Biotechnology Co., LTD. (Beijing, China). AMPK, PGC1-α, TFAM, NRF1 and GAPDH were purchased from Proteintech Group, Inc. (Rosemont, USA). Primers were synthesized by Genewiz Biotechnology Co., LTD. (Jiangsu, China)

Preparation and characterization of IE-Zn and OPZ complexes

IE-Zn complexes were prepared by dissolving IE (10 mg/mL) in deionized water, mixing with ZnSO₄·7H₂O, and incubating at 45 °C for 40 min. The mixture was dialyzed (200 Da membrane) for 24 h to remove unbound zinc. Zinc and protein contents were determined using atomic absorption spectrophotometry (AAS, Hitachi, Japan) and Kjeldahl nitrogen analysis. The oyster peptide zinc complex (OPZ) was prepared similarly using oyster peptide. Structural characterization of IE-Zn was conducted using Fourier-transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy, and X-ray diffraction (XRD) to assess conformational and crystallinity changes in the peptide structure post-chelation

Molecular dynamics simulation and isothermal titration calorimetry (ITC)

In order to study the molecular recognition driving force of IE and Zn²⁺, this study used molecular dynamics (MD) simulation to simulate the structures of IE and Zn²⁺ obtained by virtual screening. The IE structure was generated by alphafold3 and MD simulation was performed by Gromacs2023.4 program under constant temperature and constant pressure and periodic boundary conditions. Amber14SB full atomic force field, TIP3P water model. In the MD simulation process, all the hydrogen bonds involved were constrained by LINCS algorithm, and the integral step was 2 fs. The electrostatic interaction is calculated using the Particle-mesh Ewald PME method. The non-bond interaction cutoff value is set to 10 A. V-rescale temperature coupling method was used to control the simulated temperature of 298.15 K, and Parrinello-Rahman method was used to control the pressure of 1 bar. The steepest descent method is used to minimize the energy of the two systems to eliminate the too close contact between atoms. Then, the NVT balance simulation of 100 ps was performed at 298.15 K. Finally, 100 ns MD simulation of the system was carried out, and the visualization of simulation results was completed by Gromacs embedded program, VMD and pymol.

For thermodynamic characterization, ITC (TA, USA) was conducted with Zn (160 μL, 5 mM) titrated into an IE solution (350 μL, 1 mM) to determine binding constants, using protocols modified from previous studies.

Cell culture and viability assay

Caco-2 and IEC-6 cells were cultured in MEM and DMEM, respectively, supplemented with FBS and penicillin–streptomycin solution. Cells were seeded in 96-well plates and treated with varying concentrations of IE-Zn and ZnSO₄ for 24 h. Viability was assessed using the CCK8 assay with absorbance measured at 450 nm.

Cell model of zinc deficiency

Zinc-deficient media were prepared by treating FBS with Chelex-100 resin to remove zinc. The method is based on Finamore [21] and Tate [22] and modified slightly. Chelex resin was added to FBS (FBS-Zn) at 10 % and stirred at 4℃ for 48 h. The content of metal ions in FBS and FBS-Zn after chelation was determined by atomic absorption spectrometry, and then sufficient metal ions (except zinc) were re-added to FBS-Zn. Table 1 shows the concentrations of various elements in both media. Two FBS were used to configure the cell culture medium

Transport analysis in Caco-2 Monolayers

Caco-2 cells were seeded at a density of 1.0 × 10⁴ cells / well in 12-well transwell plates. Transwell inserts to form monolayers and cultured for 21 days to measure transepithelial electrical resistance (TEER) and alkaline phosphatase (AKP) activity. Sodium fluorescein permeability assays verified monolayer integrity. The transport, retention and absorption of IE-Zn were assessed using these Caco-2 monolayers by applying IE-Zn solution (100 μM), to the apical (AP) side and quantifying zinc in the apical medium, cells and basolateral (BL) side via AAS. The calculation method is shown in formula (1)–(3)

$$\mathit{Transport}rate(\%)=\frac{C_{\mathit{BL}}}{C_0}\times 100$$ (1)

$$\mathit{Retention}rate(\%)=\frac{C_{\mathit{cell}}}{C_0}\times 100$$ (2)

$$\mathit{Absorption}rate(\%)=\frac{C_0-C_{\mathit{AP}}}{C_0}\times 100$$ (3)

where C₀ is the concentration of zinc ion added to the sample; C_AP is the concentration of Zn²⁺ at AP side after absorption. C_BL is the concentration of Zn²⁺ on BL side after absorption. C_cell is the concentration of Zn²⁺ in Caco-2 cells after absorption.

Pathway-specific inhibitors including ZIP4 inhibitor NFS-ZP7-4 (0.5 mM), entophagocytotic pathway inhibitor m-βcd (1 mM), and cytochalasin-D (1.5 mM) were used to determine the transport mechanisms of IE-Zn in Caco-2 cells [23]. Three inhibitors were evaluated by Caco-2 monolayer in IE-Zn solution containing 100 μM Zn²⁺.

TSQ fluorescent staining and ATP analysis

TSQ (N-(6-Methoxy-8-quinolyl)-P-Toluenesulfonamide) is a cell penetrating and highly sensitive fluorescent zinc ion probe that can detect intracellular Zn²⁺ change [24]. TSQ staining was used to visualize zinc uptake in cells treated with IE-Zn and ZnSO₄ over different time intervals, with fluorescence measured by inverted microscopy. ATP levels were quantified in treated cells using an ATP detection kit, with chemiluminescence measured to assess mitochondrial function

Flow cytometry and mitochondrial membrane potential assay

Flow cytometry (BD, USA) was used to quantify ROS and apoptosis, with cell suspensions analyzed for 10,000 cells per sample.

JC-1 was used as a fluorescent probe to detect mitochondrial membrane potential. The changes of red and green fluorescence in JC-1 staining were observed under fluorescence microscope to evaluate the changes of mitochondrial membrane potential.

Animal model of zinc deficiency and tissue analysis

Animal experiments were approved by the Dalian Polytechnic University Animal Ethics Committee (number: DLPU2024016). Four-week-old male C57 mice were purchased from Liaoning Changsheng Biotechnology Co., LTD. All mice were provided with free access to food and water. Furthermore, they were housed under the standard conditions, which are 12 h light/dark cycle at an environmental temperature of 23 ± 2 ℃ and humidity of 55 ± 5 %.

The mice were divided into two groups and administered either OPZ or ZnSO₄ (3.8 mg/Kg), with blood and tissue samples collected at predetermined intervals, including 10 min, 20 min, 40 min, 1 h, 6 h, 24 h, and 24 h. Serum, small intestine, liver, and bone samples were used to assess zinc levels and pharmacokinetic profiles.

qRT-PCR analysis

The cells (1 × 10⁶ cells/well) were seeded and cultured to mature, and treated with IE-Zn and ZnSO₄ respectively for 24 h. For qRT-PCR analysis, RNA was extracted from treated cells, converted to cDNA, and analyzed for gene expression using GAPDH as a reference. The results were calculated by 2^-ΔΔCt method. The primer sequence has shown in Table 2.

Western blotting was conducted on cell lysates to detect proteins involved in zinc transport (ZIP4, ZNT1, DMT1) and energy metabolic pathway (AMPK, PGC1-α, NRF-1, TFAM) with protein bands visualized on a Bio-Rad chemiluminescence system.

Statistical analysis

Data are presented as mean ± SD unless indicated otherwise. For all experiments, three technical replicates were performed. The difference analysis for multiple groups was assessed by one-way ANOVA, Tukey’s multiple comparisons test and Dunn’s multiple comparisons tests. The GraphPad Prism 10.0.3 (La Jolla, Canada) was employed to process the figures and perform statistical analysis. Fluorescence intensity and gray value of protein band were measured by Image J software (National Institute of Health, USA). GROMACS was used for molecular dynamics simulation of IE and Zn²⁺. The pymol was used to process the results of IE and Zn²⁺ molecular dynamics simulations

Results and discussion

Interaction dynamics between IE and Zn²⁺

The molecular dynamics simulations provide a detailed view of the structural relationship between IE and Zn²⁺, identifying three primary binding sites. The first zinc ion binds to E6 and E9, while the third zinc ion binds to residues at E7 and E11. The second zinc ion, located at E5, shows a comparatively weaker bond strength. These binding positions and bond lengths indicate that electrostatic interactions play a central role, particularly in stabilizing the first and third zinc ions. The shorter bond lengths of these ions, at 4.00 Å and 5.36 Å, respectively, suggest they are more structurally integral to the peptide-zinc complex, potentially reinforcing the peptide conformation to better withstand cellular environments (Fig. 1A-C). The observed secondary structure alterations highlight a transition in IE from a relatively ordered α-helix to a more disordered structure upon binding with Zn²⁺. This shift implies that the complex adopts a more flexible configuration, which may enhance its stability and adaptability in biological systems. Specifically, the reduction in α-helix content and increase in random coils (Fig. 1B) suggest that zinc binding disrupts hydrogen bonding patterns within the peptide, thereby altering its tertiary conformation. Such structural changes could enhance the bioactivity of the peptide, enabling improved interactions with cellular zinc transporters or receptors

Fig. 1.

Evaluation of interaction between IE and zinc ions. (A) Molecular dynamics simulation. (B) Secondary structure changes. (C) Energy changes. (D) Thermotitration curve of the interaction of IE and zinc ions using isothermal titration calorimetry. (E) The heat of reaction per injection (kcal/mol) was calculated and fitted using an independent binding model. Thirty drops of zinc sulfate (5 mM) were dropped into IE (1 mM) solution at 37 °C.

Isothermal titration calorimetry (ITC) analysis corroborates these findings, where the interaction between IE and Zn²⁺ was shown to be spontaneous, with a negative enthalpy change (ΔH = −21.28 kJ mol⁻¹) and positive entropy shift (ΔS = 2.171 J mol⁻¹ K⁻¹) (Fig. 1D-E). The exothermic nature of the reaction points to a highly stable complex, where the release of heat upon Zn²⁺ binding confirms the formation of strong ionic and hydrogen bonds [25]. The favorable entropy change suggests that hydrophobic interactions and structural rearrangements, such as the transition to a more flexible, random coil-dominated structure, are integral to the binding process. This entropy-driven reaction is indicative of zinc’s affinity for forming multiple, less rigid associations with the peptide, thereby enabling dual-specific and non-specific binding. The binding constant (K_d = 144 μM⁻¹) and n number of approximately 2.062 reflect a high-affinity interaction between IE and Zn²⁺, suggesting that the complex forms with both specificity and stability. Such an affinity indicates that, at physiological zinc concentrations, IE can efficiently chelate Zn²⁺ ions. This could have significant implications for its bioavailability and the bioaccessibility of zinc in biological systems. In fact, the specific and non-specific binding modes observed may allow the peptide complex to maintain zinc binding under diverse physiological conditions, potentially contributing to its enhanced absorption capabilities as demonstrated in subsequent cellular transport experiments.

These findings provide insight into how IE-Zn complexes could serve as a superior zinc supplement, with specific molecular interactions that confer stability, flexibility, and potentially enhanced bioactivity. The dual binding modes, specific electrostatic interactions coupled with more versatile hydrogen bonding, may enable the IE-Zn complex to exhibit robust absorption characteristics. This unique binding profile distinguishes IE-Zn from ZnSO₄, and supports its potential as a targeted, bioavailable zinc source with structural advantages that can influence its uptake and function in vivo.

Structural characterization and implications for IE-Zn complex functionality

The binding interaction between Zn²⁺ and the functional groups within the IE peptide, as evidenced by FT-IR (Fig. 2A) and XRD (Fig. 2B), suggests a fundamental shift in the peptide's stability and conformational profile. The –NH absorption peak moves from 3286.64 cm⁻¹ to 3277.91 cm⁻¹ [26]. The −N–H bond vibrates when Zn²⁺ binds to IE, and Zn²⁺ replaces hydrogen atoms [27]. The absorption peak of COO− shifts from 1410.74 cm⁻¹ to 1406.41 cm⁻¹, and Zn ²⁺ reacts with COO− to form COO-Zn [28]. The spectral band of amide I of the peptide molecule moved from 1628.90 cm⁻¹ to 1629.12 cm⁻¹ [29]. In summary, combined with the molecular dynamics simulation results, Zn²⁺ mainly binds to the carboxyl group, resulting in stable electrostatic interactions and resonance throughout the peptide chain. This form of coordination is indicative of a strengthened internal framework that can better withstand the acidic and enzymatic environment of the digestive tract, thus reducing the rate of premature degradation. Such stability is further corroborated by the XRD analysis, where the shift toward a semi-crystalline state upon Zn²⁺ incorporation highlights a structural refinement within the peptide matrix, which was consistent with Wang et al. [16] These structural modifications suggest that Zn²⁺ ions serve as a stabilizing force, enhancing the peptide's resilience and ultimately contributing to its sustained bioavailability

Fig. 2.

Changes in the apparent characteristics of IE and IE-Zn. (A) Fourier transform infrared spectra (FTIR) of IE and IE-Zn in the range of 4000–400 cm⁻¹. (B) X-ray diffraction (XRD) detects diffraction patterns of 5–90^◦ to detect changes in the crystal structure of the resulting sample. (C, D) CD spectra and secondary structure of IE and IE-Zn.

While the IE-Zn complex demonstrates increased stability, CD spectroscopy reveals that the peptide's secondary structure becomes more flexible due to Zn2⁺ binding, with an observed increase in random coil content (Fig. 2C-D). This may be mainly due to the addition of zinc weakening the hydrogen bonds and electrostatic interactions of the alpha-helix and β-sheets, and the protein structure becoming looser with the increasing of random coils [30]. This conformational flexibility could facilitate interactions with cellular receptors, transporters, and zinc uptake mechanisms within intestinal epithelial cells. The shift from rigid α-helical and β-sheet structures to a more dynamic, coil-rich structure implies that the IE-Zn complex can better conform to various cellular environments, thereby potentially improving its transport and absorption efficiency. This flexibility is crucial, as it suggests the complex can readily adapt to the conformational requirements of different cellular transport pathways, particularly those that are not accessible to more rigid zinc sources, such as ZnSO₄.

The spectroscopy results together provide a comprehensive understanding of how Zn²⁺ binding contributes to the peptide’s structural integrity and functional flexibility. The combined findings suggest that the IE-Zn complex presents a zinc source with enhanced bioavailability, characterized by both structural stability and adaptability. This combination aligns well with the biological goals of zinc supplementation. By stabilizing the peptide through Zn2⁺ coordination, the complex effectively shields zinc ions from premature degradation while simultaneously promoting efficient cellular transport. These properties set it apart from conventional zinc sources, such as ZnSO₄.

Enhanced zinc absorption efficiency of IE-Zn compared to ZnSO₄

Transport studies using the Caco-2 cell model reveal that the IE-Zn complex exhibits a significantly higher zinc uptake rate than ZnSO₄ under similar conditions, highlighting the structural and binding advantages of the IE-Zn complex for zinc bioavailability. TEER, AKP assays and translocation of sodium fluorescein confirmed the integrity of the Caco-2 monolayer and differentiation degree [31], with TEER values consistently exceeding 600 Ω·cm², AKP activity peaking at 20 days and the sodium fluorescein transmittance of blank control group was 4 times that of the Caco-2 cell group (Fig. 3A-D). These results establish the reliability of the Caco-2 model as a proxy for human intestinal absorption, allowing for the analysis of zinc dynamics across an epithelial barrier [32] and providing insights into how IE-Zn interacts with this cellular environment

Fig. 3.

Intestinal transport and absorption of zinc ions in IE-Zn and ZnSO₄. (A) The Transwell system mimics absorption in the human small intestine. (B) Transepithelial electric resistance (TEER) measurements were conducted on monolayer Caco-2 cells during their differentiation process for a total of 21 days (n = 12). (C) Relationship between AKP monolayer and growth time of Caco-2 cells. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Tukey's multiple comparisons test was performed. Different letters represent significant differences p < 0.05. (D) The transmittance of sodium fluorescein through Transwell plate (21th). n = 3. Graphs represented the mean ± SD. Two-way ANOVA followed by Šídák's multiple comparisons test was performed, **** p < 0.0001 versus control group. (E-G) Effects of IE-Zn and ZnSO₄ on zinc transport rate, retention rate and absorption rate of Caco-2 cells. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Tukey's multiple comparisons test was performed, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control group.

Comparative absorption studies revealed that the transport rates of IE-Zn and ZnSO₄ were not significantly different. However, IE-Zn complex led to a significantly higher rate of Zn²⁺ uptake, reaching 77.3 % at the 2-hour mark, compared to ZnSO₄, which only achieved an 63.1 % absorption rate under identical conditions (Fig. 3E-F). This finding suggests that the peptide-bound zinc in IE-Zn is released in a sustained manner, likely due to shielding effects of the peptide structure, which may prevent immediate release or interactions with competing dietary components. In addition, the retention rate for IE-Zn-treated cells was 53.9 %, contrasting with a 40.9 % retention rate for ZnSO₄ (Fig. 3G). This suggests a more gradual release profile for IE-Zn, as the peptide structure enables zinc retention, enhancing its cellular utilization over time, which is consistent with the results of Wang et al. [16]. Further support for these findings comes from the TSQ fluorescence assay, where IE-Zn had a stronger fluorescence intensity than ZnSO₄ at 1-hour labeling, indicating a higher intracellular zinc concentration (Fig. 4A, S1). These results suggest that IE-Zn engages multiple cellular uptake pathways, unlike ZnSO₄, which primarily relies on ZIP transporters. By contrast, IE-Zn appears capable of leveraging both metal-ion transporters and peptide pathways, a hypothesis supported by the upregulation of PEPT1 expression in IE-Zn-treated cells (Fig. S2A). This dual pathway of absorption likely allows IE-Zn to bypass the saturation limits of ZIP-mediated transport, contributing to enhanced zinc bioavailability. Additionally, by measuring the small intestine of mice ingested with OPZ and ZnSO₄, the increased expression of Zip4, Znt1 and Pept1 in the IE-Zn group further suggests that the peptide-bound form of zinc may facilitate multi-transporter engagement, thus promoting zinc uptake more effectively than ionic zinc alone (Fig. 5B-D).

Fig. 4.

Evaluation of zinc ion transport of IE-Zn in Caco-2 cells and pharmacokinetics. (A) Evaluation of zinc ion absorption efficiency of ZnSO₄ and IE-Zn in different time periods. Zinc ion fluorescent probe in Caco-2 cells were assessed after 5 min, 10 min, 20 min, 40 min and 60 min. n = 3. (B) Timeline of mice sampling in pharmacokinetics. (C-F) Zinc ion content in serum, small intestine., liver and shank. n = 3. Graphs represented the mean ± SD. Two-way ANOVA followed by Sidak's multiple comparisons test was performed, * p ≤ 0.05, ** p < 0.01, *** p < 0.001, and **** p ≤ 0.0001 between two groups.

Fig. 5.

The absorption mechanism of zinc ions in IE-Zn and ZnSO₄. (A) Transport diagram of zinc ion and peptide zinc complex. (B-D) The mRNA expressions of ZIP4, ZNT1 and PEPT1 in the small intestine of mice in pharmacokinetic experiments. n = 3. Two-way ANOVA followed by Sidak's multiple comparisons test was performed, * p ≤ 0.05, ** p < 0.01, *** p < 0.001, and **** p ≤ 0.0001 between two groups. (E-G) The mRNA expressions of ZIP4, ZNT1 and DMT1 in Caco-2 cells were assessed after 24 h of intervention with IE-Zn and ZnSO₄. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Dunnett's multiple comparisons test was performed, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control group. (H-K) The protein expressions of ZIP4, ZNT1 and DMT1 in Caco-2 cells were assessed after 24 h of intervention with IE-Zn and ZnSO₄. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Dunnett's multiple comparisons test was performed, * p < 0.05 versus control group.

The pharmacokinetic analysis in mice aligns well with the in vitro results, showing elevated zinc levels in serum, small intestine, and liver after administration of IE-Zn, compared to ZnSO₄. In serum, IE-Zn achieved a zinc concentration peak of 66.5 μg/mL at 40 min post-administration, while ZnSO₄ peaked at 44.4 μg/mL (Fig. 4C). Similarly, in the small intestine, IE-Zn reached 190.5 μg/g, compared to 174.7 μg/g for ZnSO₄ (Fig. 4D). These elevated levels in vivo indicate that the IE-Zn complex is not only absorbed more efficiently in the intestinal tract but also distributed more effectively throughout the system [33]. The peptide-bound form's stability likely prevents the rapid clearance that ZnSO₄ undergoes, allowing IE-Zn to provide a sustained zinc release, with the peptide ligands potentially stabilizing zinc ions against rapid degradation or binding within the gastrointestinal environment [34].

The combined results from these studies underscore the unique capabilities of IE-Zn, which appears to operate through a dual absorption mechanism, combining the strengths of both ionic and peptide-bound zinc transport. This layered absorption profile not only enhances the efficiency of zinc uptake but also suggests that IE-Zn could better modulate cellular zinc homeostasis than ZnSO₄. IE-Zn's multi-transporter absorption mechanisms allow it to function as a more robust zinc delivery system, demonstrating potential as an advanced zinc supplement suitable for therapeutic applications where optimized zinc bioavailability is necessary.

Mechanistic insights into IE-Zn transport and absorption

In this study, the IE-Zn complex was shown to facilitate Zn²⁺ absorption through multiple pathways, resulting in enhanced zinc bioavailability and influencing cellular zinc homeostasis. ZIPs introduce zinc ions from the extracellular space or intracellular vesicles into the cytoplasmic compartment to supplement the transport and absorption proteins of zinc ions in the cytoplasm. ZNTs are efflux transporters that export excess zinc ions from the cytoplasmic compartment to the extracellular space or intracellular vesicles [35]. DMT1 transports bivalent cations in exchange for a single proton, mediating the transport of essential and toxic bivalent metal ions, including Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Co²⁺ and Cd²⁺ [36]. Zinc ions are absorbed by passive transport (alternative and endocytic pathways) and active transport, of which active transport is particularly important. The active transport of zinc in intestinal epithelial cells can be divided into three steps. First, zinc ions enter the intestinal epithelial cells from the intestinal cavity via membrane transporters (mainly ZIP4 and DMT1), then zinc ions move from the upper part of the intestinal epithelial cells to the lower part via MT or free diffusion, and finally zinc ions enter the blood via membrane transporters (such as ZNTs) [37]. The complementary function of ZNTs and ZIPs stabilizes cytoplasmic zinc concentrations while enriching zinc in specific subcellular compartments to support zinc-dependent cellular processes [38]. The increased expression of zinc transporters, such as ZIP4 and ZNT1, in both Caco-2 cells and in vivo models treated with IE-Zn demonstrates this dual absorption mechanism. Specifically, the mRNA levels of ZIP4 were observed to increase by approximately 1.87-fold in Caco-2 cells treated with IE-Zn compared to cells treated with ZnSO₄ (Fig. 5E). This result suggests that peptide-bound zinc induces more efficient cellular uptake by stimulating specific zinc transporter pathways. Previous studies have indicated that ZIP4 plays a critical role in the absorption of dietary zinc across the apical membrane of enterocytes, thus supporting the hypothesis that IE-Zn utilizes these transporters more effectively due to its distinct binding properties. Moreover, a substantial upregulation of PEPT1 expression by 1.86-fold relative to the control was observed in IE-Zn-treated Caco-2 cells, DMT1 and MT1 did not increase significantly, highlighting the potential of the zinc-peptide complex to utilize oligopeptide transporters in addition to traditional zinc ion channels (Fig. 5G, S2A-B). The engagement of PEPT1 suggests that IE-Zn may be absorbed as a peptide complex, which subsequently dissociates intracellularly, allowing for a dual route of zinc delivery. This mechanism sets IE-Zn apart from ZnSO₄, which relies solely on ion transport channels, as demonstrated by the lack of significant PEPT1 activation in ZnSO₄-treated cells. The ability of IE-Zn to interact with both metal-ion and peptide transport pathways is consistent with other research on zinc-bound peptides, indicating that such complexes can facilitate zinc’s homeostasis and bioavailability by preventing premature dissociation and enabling efficient transport across membranes

Considering the absorption and transport process, it is not only a mode of transmembrane transport. Further investigation using specific inhibitors of active transport (NVS-ZP7-4), endocytosis (m-βcd), and paracellular transport (cytochalasin D) revealed that IE-Zn utilizes multiple pathways for transport. A decrease of approximately 35 % in zinc transport efficiency was observed when active transport pathways were inhibited, while endocytic and paracellular pathways also showed significant reductions (Fig. S3). These results indicate that IE-Zn does not rely on a single absorption mechanism. It employs a flexible strategy that optimizes bioavailability by engaging various transport routes, which may provide an advantage under varying physiological conditions. This multifunctional uptake mechanism may arise from the peptide’s structural properties, which stabilize zinc and facilitate its passage through different transporter types, as suggested by the structural analyses presented earlier in this study. Wang found that the transport pathway of a new Novel Casein-Derived Peptide-Zinc Chelate TEDELQDKIHP-Zn also had similar results [39]. The dual pathway engagement of IE-Zn holds broader implications for zinc homeostasis. Upon cellular entry, zinc appears to stimulate a regulatory feedback mechanism involving the upregulation of ZNT1, an efflux transporter that helps balance intracellular zinc levels. The ZNT1 mRNA and protein levels were observed to increase by 3.74-fold and 2.61-fold in cells treated with IE-Zn compared to ZnSO₄, suggesting a coordinated cellular response aimed at redistributing zinc within the cytoplasm to avoid potential cytotoxicity (Fig. 5E, H, K). This finding aligns with studies on zinc homeostasis that have documented similar regulatory adjustments in response to elevated intracellular zinc, where ZNT family members function to sequester excess zinc in vesicles or facilitate its export from the cell.

These findings underscore the superior absorption profile of IE-Zn relative to ZnSO₄. The complex's ability to engage with both zinc ion and oligopeptide pathways not only enhances zinc uptake efficiency but also supports a more balanced intracellular distribution [40]. This dual transport mechanism suggests that IE-Zn could serve as an effective zinc supplement, especially for individuals with compromised zinc absorption or increased zinc requirements. Furthermore, the peptide complex’s interactions with multiple transporter systems provide a potential therapeutic avenue for applications requiring targeted zinc delivery. Overall, the multifunctional absorption strategy of IE-Zn supports its suitability as a bioavailable zinc source, with structural and functional advantages that distinguish it from traditional inorganic zinc salts, as evidenced by the improved zinc transport, cellular uptake, and homeostatic response observed in this study.

IE-Zn mitigates zinc deficiency-induced energy metabolism disturbances in Caco-2 cells

Zinc plays an essential role in maintaining cellular energy homeostasis, zinc deficiency will cause the disturbance of energy metabolism[41]. In zinc-deficient Caco-2 cells, the disruptions in energy metabolism were significantly reversed by IE-Zn supplementation. Notably, IE-Zn treatment increased AMPK mRNA and protein expression by approximately 1.65-fold and 1.60-fold relative to the zinc-deficient control, while ZnSO₄ produced a more modest 1.10-fold and 1.22-fold increase (Fig. 6A, E, I). AMP-activated protein kinase (AMPK) is a signal-modulating integrator that regulates the energy state of systems and cells in response to environmental nutrient changes. Disruption of AMPK pathway leads to metabolic dysregulation, leading to various metabolic disorders [42]. This finding indicates that IE-Zn enhances cellular energy regulation pathways more robustly than ZnSO₄. The zinc-peptide complex appears to optimize zinc utilization within these metabolic pathways, suggesting a structural advantage in supporting cellular energy balance

Fig. 6.

The regulating effect on AMPK/PGC1-α/NRF-1/TFAM signal axis in Caco-2 cells. (A-D) The mRNA expressions of AMPK, PGC-α, NRF-1 and TFAM in Caco-2 cells were assessed after 24 h of intervention with IE-Zn and ZnSO₄. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Dunnett's multiple comparisons test was performed, * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control group. (E-I) The protein expressions of AMPK, PGC1-α, NRF-1 and TFAM in Caco-2 cells were assessed after 24 h of intervention with IE-Zn and ZnSO₄. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Dunnett's multiple comparisons test was performed, * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control group.

The AMPK/PGC1-α signaling axis is a critical pathway that regulates cellular energy balance and mitochondrial biogenesis, making it a key target for therapies addressing metabolic stress. PGC1-α (peroxisome proliferator-activated receptor gamma coactivator 1-α) is a major regulator that plays an important role in cellular energy metabolism and mitochondrial biogenesis [43]. In parallel, the mitochondrial biogenesis regulator PGC1-α was upregulated by 1.8-fold in response to IE-Zn compared to the zinc-deficient group, with ZnSO₄ showing a less pronounced effect (1.2-fold increase) (Fig. 6B). At the protein level, IE-Zn also promoted PGC1-α expression more effectively, likely aiding mitochondrial adaptation to metabolic demands (Fig. 6F, I). Activation of AMPK triggers downstream effects, including the upregulation of PGC1-α, which drives mitochondrial biogenesis and energy production. Similarly, the transcription factor NRF-1 was upregulated by 1.6-fold with IE-Zn, whereas ZnSO₄ resulted in only minor increases (Fig. 6C). NRF-1 promotes mitochondrial synthesis and function by activating genes that code for mitochondrial proteins. This suggests that IE-Zn enhances mitochondrial gene expression through more comprehensive support of zinc-dependent transcriptional machinery, which may facilitate better mitochondrial function and resilience. As a critical protein for mitochondrial DNA replication and transcription [44]. TFAM exhibited a 2.3-fold increase with IE-Zn supplementation, in contrast to a 1.7-fold increase with ZnSO₄ (Fig. 6C, G, I). Elevated TFAM levels point to improved mitochondrial stability, driven by the enhanced transcriptional capacity provided by IE-Zn. The complex structure of IE-Zn may allow more efficient zinc transport into mitochondria, thereby supporting the organelle's function. Consequently, ATP production in the IE-Zn treated cells increased by 97 %, substantially more than the 70 % rise observed with ZnSO₄ (Fig. 7F). This higher ATP yield implies a more robust energy metabolism in cells treated with IE-Zn, possibly due to the combined effects of improved zinc absorption and enhanced mitochondrial function.

Fig. 7.

IE-Zn alleviates mitochondrial damage caused by zinc deficiency. (A-B) Apoptosis changes in cells subjected to zinc deficiency and administration of zinc ions. The number of cells cultured in normal medium and zinc-deficient medium for two days. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Dunnett's multiple comparisons test was performed, ** p < 0.01 and **** p < 0.0001 versus −Zn group. (C-E) Changes in ROS levels. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Dunnett's multiple comparisons test was performed, **** p < 0.0001 versus −Zn group. (F) Changes in ATP content. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Dunnett's multiple comparisons test was performed, *** p < 0.001, and **** p < 0.0001 versus −Zn group. (G) The number of cells cultured after two days in a zinc-deficient environment. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Dunnett's multiple comparisons test was performed, **** p < 0.0001 versus −Zn group. (H) Schematic diagram of JC-1 fluorescence change. (I-J) Mitochondrial membrane potential changes of IE-ZN and ZnSO₄ in a zinc-deficient environment. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Dunnett's multiple comparisons test was performed, *** p < 0.001 versus −Zn group.

The dual regulatory effect of IE-Zn on cellular energy metabolism suggests both direct and indirect benefits. By directly supplying bioavailable zinc and indirectly activating key metabolic pathways, IE-Zn appears to improve cellular adaptation to energy stress. The zinc-peptide complex likely modulates zinc transport more effectively, activating AMPK and downstream effectors like PGC1-α and TFAM more efficiently than ZnSO₄. This effect aligns with recent studies suggesting that peptide-bound zinc forms can exhibit superior cellular uptake and signaling potential over simple inorganic zinc salts. Overall, IE-Zn significantly mitigates zinc deficiency-induced energy metabolism disruptions. Its interaction with the AMPK/PGC1-α/NRF-1/TFAM axis restores cellular energy balance [45], suggesting that IE-Zn can serve as a highly bioavailable zinc supplement with therapeutic potential. By promoting mitochondrial biogenesis and function, IE-Zn could offer distinct advantages in managing conditions characterized by impaired energy metabolism and zinc deficiency. These findings underscore the broader utility of zinc-peptide complexes in supporting metabolic health.

Protective effects of IE-Zn on mitochondrial integrity and oxidative stress

It is found that zinc, as an important trace element in the human body, plays an important role in energy metabolism. Apoptosis is a vital part in the life activities of multicellular organisms, and zinc deficiency affects the process of apoptosis. The process of apoptosis is also very complex, in which oxidative stress can lead to mitochondrial dysfunction, including the destruction of mitochondrial membrane potential. Zinc-deficient cells showed an apoptosis rate of approximately 10.7 %, whereas IE-Zn-treated cells exhibited a significantly reduced rate of around 6.2 %, almost returning to baseline. In contrast, ZnSO₄ reduced apoptosis to only 9.1 %, which, while beneficial, was less effective than IE-Zn (Fig. 7A-B). These results highlight the enhanced protective role of IE-Zn against zinc-deficiency-induced cellular apoptosis

Oxidative stress due to zinc deficiency was associated with increased apoptosis and mitochondrial dysfunction in IEC-6 cells. ROS levels as measured by flow cytometry, were similarly impacted. Upon zinc deficiency, ROS levels increased by over 1.83-fold compared to the control, indicating significant oxidative stress. However, with IE-Zn supplementation, ROS levels decreased by nearly 27.4 % relative to the zinc-deficient group, while ZnSO₄ only achieved a 13.6 % reduction (Fig. 7C-E). This finding suggests that IE-Zn more effectively mitigates oxidative stress than ZnSO₄.

In addition to reducing oxidative stress and apoptosis, IE-Zn treatment stabilized mitochondrial membrane potential (ΔΨm). the decrease in mitochondrial membrane potential is also a landmark indicator of early apoptosis [46]. The JC-1 assay demonstrated that, in zinc-deficient conditions, mitochondrial depolarization occurred, evidenced by a high green-to-red fluorescence ratio. With IE-Zn supplementation, the fluorescence ratio improved by 7.57-fold, while ZnSO₄ led to a 2.09-fold improvement (Fig. 7H-J). These results are consistent with those of ATP (Fig. 7F), suggesting that IE-Zn can effectively protect mitochondrial integrity and promote mitochondrial health compared with ZnSO₄. Meanwhile, the expression levels of key mitochondrial markers in the AMPK/PGC1-α/NRF-1/TFAM axis was also enhanced. IE-Zn increased Ampk mRNA expression by 2.11-fold, Pgc1-α by 1.82-fold, and Tfam by 23.9-fold, relative to the zinc-deficient condition (Fig. 8A-D). In contrast, ZnSO₄ treatment led to a lesser increase across these markers, suggesting that IE-Zn may play a more pronounced role in supporting mitochondrial biogenesis and energy regulation. Nrf-1 protein levels increased by approximately 1.66-fold in IE-Zn-treated cells (Fig. 8G, I). These results indicate that IE-Zn not only supports mitochondrial function but also promotes enhanced mitochondrial biogenesis.

Fig. 8.

The regulating effect on Ampk/Pgc1-α/Nrf-1/Tfam signal axis in IEC cells. (A-D) The mRNA expressions of Ampk, Pgc1-α, Nrf-1 and Tfam in IEC cells were assessed after 24 h of intervention with IE-Zn and ZnSO₄. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Dunnett's multiple comparisons test was performed, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus −Zn group. (E-I) The protein expressions of Ampk, Pgc1-α, Nrf-1 and Tfam in IEC cells were assessed after 24 h of intervention with IE-Zn and ZnSO₄. n = 3. Graphs represented the mean ± SD. One-way ANOVA followed by Dunnett's multiple comparisons test was performed, * p < 0.05, versus −Zn group. (J) Schematic diagram of AMPK/PGC1-α/NRF-1/TFAM signal axis of energy metabolism.

Collectively, these data indicate that IE-Zn mitigates oxidative stress and supports mitochondrial function more effectively than ZnSO₄. By restoring mitochondrial integrity and improving antioxidant defenses, IE-Zn has a dual role in both cellular protection and metabolic support. The superior activation of AMPK and downstream effectors (PGC1-α, NRF-1, and TFAM) by IE-Zn underscores its ability to restore energy homeostasis and promote mitochondrial biogenesis through a multi-targeted mechanism (Fig. 8J). These effects highlight the potential of peptide-zinc complexes as a bioavailable zinc supplement with enhanced therapeutic benefits over traditional zinc salts, aligning with findings that emphasize the need for improved zinc delivery systems in conditions of high oxidative stress and compromised mitochondrial function.

Conclusion

This study demonstrates that the IE-Zn complex offers significant advantages over traditional zinc supplements like ZnSO₄, particularly in terms of bioavailability, cellular absorption, and metabolic impact. Through a combination of specific binding mechanisms, involving both electrostatic and hydrogen bonding, IE-Zn shows a stable and flexible structure that facilitates zinc transport via dual pathways, which is supported by increased expression of PEPT1 in Caco-2 cells. These properties contribute to higher absorption and retention rates, highlighting IE-Zn’s potential as a targeted zinc supplement. Additionally, the IE-Zn complex activates the key energy-regulating pathways (AMPK/PGC1-α/NRF-1/TFAM), promoting mitochondrial biogenesis and ATP production, and reducing oxidative stress markers such as ROS, thereby mitigating the effects of zinc deficiency on energy metabolism. The findings indicate that IE-Zn could play a significant role in addressing conditions associated with mitochondrial dysfunction and zinc deficiency. Future research could focus on the development of functional foods for special populations, and potential prophylactic applications for populations in need. I think this is not limited to zinc deficiency people, and even some people with zinc deficiency related to mental malaise, intestinal inflammation and skin diseases. These insights lay the groundwork for further exploration of bioactive peptide-mineral complexes as advanced supplements with multifunctional health benefits.

Compliance with Ethics Requirements

All Institutional and National Guidelines for the care and use of animals (fisheries) were followed.

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

The following are the Supplementary data to this article: