The effect of liposomal ellagic acid on the biological properties of dental restorative materials: An in vitro comparative study

Abstract

This study investigated whether incorporating liposomal ellagic acid (LEA) could enhance the biological performance of two common dental restorative materials. LEA nanoparticles were synthesized and applied (10 μg/mL) to nano-glass ionomer and nano-dental cement discs. Human gingival fibroblasts were used to assess cytocompatibility, cell adhesion, inflammatory cytokine production, and oxidative stress. LEA-modified materials maintained over 96% cell viability, increased adhesion by 1.3-fold at day 3, and markedly reduced IL-6 and TNF-α levels compared to unmodified controls. Oxidative stress markers, including reactive oxygen species and malondialdehyde, were significantly decreased in treated groups. A sustained release profile (56% at 72 h) confirmed stable delivery from the restorative matrix. These findings suggest that LEA incorporation may enhance the antioxidant and anti-inflammatory profile of restorative materials while enhancing biocompatibility, supporting its potential as a bioactive additive for next-generation therapeutic restoratives.

Highlights

• •LEA-modified dental restoratives enhance HGF viability and adhesion.
• •Liposomal delivery enables sustained release of ellagic acid over 72 h.
• •LEA reduces ROS and MDA levels, mitigating oxidative stress in vitro.
• •Anti-Inflammatory effects shown by decreased IL-6 and TNF-α secretion.
• •First use of drug-loaded liposomes to improve restorative material biocompatibility.

1. Introduction

Dental restorative materials such as nano-glass ionomers (NGIs) like Fuji IX GP Extra and nano-dental cements (NDCs) like Biodentine are widely used for their mechanical strength, handling ease, and bioactive potential. Fuji IX offers sustained fluoride release, while Biodentine provides strong sealing and promotes pulp and dentin repair [[1], [2], [3]]. However, both face significant biological limitations, including cytotoxicity, oxidative stress, and pro-inflammatory responses, that can compromise restoration longevity.

Biodentine may reduce cell viability and adhesion at high concentrations due to elevated pH and excessive ion release [4], and it has been linked to increased ROS production [5], pro-inflammatory cytokine secretion, and limited pulp regeneration [6,7]. Similarly, Fuji IX can release residual monomers such as hydroxyethyl methacrylate, triggering cytotoxic and inflammatory responses even at low concentrations, as well as elevating ROS levels [2,8,9]. These effects, confirmed across multiple studies, highlight persistent challenges in the biocompatibility of NGI and NDC, underscoring the need for restorative materials that combine mechanical performance with enhanced biological compatibility. Several recent studies have explored the modification of conventional glass ionomer cements using nanomaterials to overcome their inherent mechanical limitations [[10], [11], [12]].

Nanoparticle-based modifications have shown promising results in dentistry, with enhancing the antibacterial activity, pH stability, and cytocompatibility of intracanal medicaments [13]. Such findings support the growing interest in nano-enabled strategies to improve the biological performance of dental materials. One promising strategy to overcome these limitations is the use of liposomal carriers, which can encapsulate and protect bioactive compounds, enabling controlled release at the restoration site while improving adhesion and reducing microbial infiltration [[14], [15], [16]]. In dentistry, their applications range from caries and periodontitis treatment to targeted antimicrobial delivery and biomimetic enamel remineralization [17,18]. However, reports of liposome-modified restorative or endodontic materials are scarce, with only one study in endodontics using drug-free liposomes to alter physical properties [14]. Incorporating active agents into liposomes could extend these benefits to biological performance.

Conventional systemic drug delivery in dentistry often requires high doses to achieve therapeutic effects, increasing the risk of resistance, toxicity, and adverse reactions [19,20]. Designing bioactive materials that ensure localized, sustained release of pharmacologically active compounds via biocompatible carriers can enhance efficacy while reducing dosage and side effects [21,22].

Polyphenols are particularly promising candidates due to their antimicrobial, antioxidant, and anti-inflammatory activities, as well as their ability to promote extracellular matrix cross-linking and improve biomaterial mechanics [[23], [24], [25], [26]]. Among these, ellagic acid (EA) is notable for its potent antioxidant and anti-inflammatory effects, though its poor solubility, rapid metabolism, and low bioavailability have limited its dental applications [21,22,27]. Liposomal encapsulation (LEA) can overcome these limitations by improving EA stability, bioavailability, and controlled release [28].

This study evaluated LEA as a bioactive modifier for two restorative material classes using human gingival fibroblasts to assess oxidative stress, inflammatory responses, and cell adhesion. We hypothesized that LEA would enhance biocompatibility by reducing ROS and pro-inflammatory cytokine production while promoting cell growth and attachment, thus offering a universally applicable strategy to improve restorative material performance.

2. Materials and methods

2.1. Specimen preparation for restorative materials

Ring molds (6 mm × 3 mm) were lubricated with 5% Luwax (Sigma-Aldrich, USA). Nano-glass ionomer (Fuji IX GP Extra) and nano-dental cement (Biodentine) were prepared by mixing powder and liquid per manufacturer instructions (Table 1). The paste was packed into molds, cured at room temperature (Fuji IX: 2–3 min, Biodentine: 12 min), removed, and polished sequentially with 1200- and 4000-grit sandpaper (3 M, USA) for 20 s each. Specimens were stored vertically in 50 mL distilled water (20 °C, 24 h, dark) before use.

Table 1.

The composition and manufacturers of dental materials.

Product/manufacturer	Composition
Biodentine®/Septodont, St. Maur des Fosses, FRANCE	Powder: tricalcium silicate (80.1%), dicalcium silicate, calcium carbonate (14.9%), iron oxide, and zirconium oxide (5%). Liquid: water, calcium chloride, and partially modified polycarboxylate
Fuji IX GP Extra; GC Corporation, Tokyo, Japan	Powder: fluoroaluminosilicate glass (97%) and polyacrylic acid (dry form, 3%). Liquid: distilled water, polyacrylic acid, and tartaric acid.

2.2. Liposome preparation and characterization

Dipalmitoylphosphatidylcholine (DPPC; Avanti Polar Lipids, USA) and cholesterol (Sigma-Aldrich, USA) were dissolved in chloroform:methanol (2:1) at a 7:3 M ratio. Ellagic acid (EA; Sigma-Aldrich, USA) was added at a 10:2 M ratio. Solvent was evaporated under reduced pressure (332 mbar, 50 °C, 15 min) to form a lipid film, hydrated with PBS (final lipid: 65 mg/mL; EA: 15 mg/mL), sonicated (10 min), centrifuged (15,000 rpm, 30 min), and extruded through 100 nm polycarbonate membranes × 10 cycles.The size and polydispersity index (PDI) of the liposomes were measured using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) and the dynamic light scattering (DLS) technique. The morphology of the liposomes was examined using a transmission electron microscope (TEM; JEOL, Tokyo, Japan). For this, the samples were placed on a carbon-coated copper grid (Ted Pella, Redding, CA, USA), and images were recorded with a high-resolution TEM microscope operating at 200 kV.

Size and polydispersity index (PDI) were measured by dynamic light scattering (Zetasizer Nano ZS, Malvern, UK). Morphology was examined by transmission electron microscopy (JEOL, Japan) using carbon-coated copper grids. Encapsulation efficiency (EE%) was determined by dissolving liposomes in DMSO and measuring absorbance at 340 nm against an EA standard curve. Release kinetics were assessed by dialysis in distilled water, sampling at 1–72 h, and measuring absorbance at 330 nm.

2.3. Cell culture

Human gingival fibroblasts (HGF-1; Pasteur Institute, Iran) were cultured in DMEM (Denazist, Iran) with 10% FBS (Sigma), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C, 5% CO₂. Third-passage cells were used.

2.4. Biocompatibility assessment

HGFs (2.4 × 10⁴ cells/cm²) were treated with LEA (1–25 μg/mL, diluted in medium) for 24–72 h to determine optimal non-cytotoxic concentration (10 μg/mL). For biocompatibility, cells were seeded onto NGI, NDC, NGI + LEA, NDC + LEA, or control surfaces; viability was measured by MTT on days 1–3.

2.5. Cell adhesion

HGFs (2.4 × 10⁴ cells/cm²) were seeded onto discs; adhesion was quantified on days 1–3 by crystal violet staining (0.1%, 20 min), washing, and dye solubilization in 10% acetic acid; absorbance was read at 570 nm.

2.6. Anti-inflammatory effects

HGFs were cultured on discs for 72 h; supernatants were collected, centrifuged, and stored at −80 °C. IL-6 and TNF-α levels were quantified using ELISA kits (R&D Systems, USA).

2.7. Oxidative stress assessment

ROS levels were measured using DCFH-DA (10 μM, 30 min incubation), fluorescence at 485/530 nm normalized to protein (BCA assay). Lipid peroxidation was measured by TBARS assay (Cayman Chemical, USA); lysates were reacted with SDS and TBA reagent, heated (95 °C, 60 min), and absorbance read at 532 nm.

2.8. Statistical analyze

Data are mean ± SD from ≥3 experiments. Two-way ANOVA (viability, adhesion) or one-way ANOVA (cytokines, oxidative stress) with Tukey's test were used; significance set at p < 0.05.

3. Results

3.1. Characterization of LEA

The size and morphology of LEA were characterized using TEM and DLS techniques, as shown in Fig. 1a–b. The DLS analysis revealed that the liposomes had a size distribution ranging from 80 to 150 nm, with an average diameter of 102.74 + 11.67 nm. The PDI of the LEA was 0.11, indicating high homogeneity and stability of the formulation. The TEM images further confirmed the liposome's spherical morphology and nanoscale size, with the observed sizes consistent with the DLS results. Additionally, the EE of EA in the liposomes was determined to be 69.7%, demonstrating the effective encapsulation of EA within the liposomal structure. The stability of the LEA was also assessed in distilled water, confirming the robustness of the formulation. As shown in Fig. 1c, the release of EA from the liposomal formulation exhibited a controlled and sustained profile over time. At 24 h, the average cumulative release of LEA reached approximately 39%, which gradually increased to 56% by 72 h. The most significant increase in EA release was observed between 12 and 24 h, with a release increase of about 14%, indicating the initial stability of the liposomes and the gradual release of LEA over time. The relatively low leakage of EA from the liposomal formulation suggests that EA is strongly associated with the liposomal structure, further confirming the stability and efficiency of the liposomal delivery system.

Fig. 1.

(a) TEM image of liposomal ellagic acid (LEA) showing spherical morphology. (b) DLS intensity distribution of particle size. (c) Cumulative EA release profile over 72 h in distilled water. (d) Heatmap showing HGF viability after exposure to LEA (0–100 μg/mL) for 24–72 h (MTT assay). (e) HGF proliferation on control, NGI, NDC, NGI + LEA, and NDC + LEA surfaces over 1–3 days (MTT assay). Statistical comparisons: $ significantly different from NGI; # significantly different from NDC (p < 0.05).

3.2. Determination of the optimal dose of LEA

The optimal concentration of LEA for subsequent experiments was determined by assessing the cytotoxicity of various concentrations (1, 5, 10, 20, 50, and 100 μg/mL) on HGFs over 1, 2, and 3 days using the MTT assay. The results demonstrated that 10 μg/mL of LEA was selected as the optimal dose due to its favorable balance between efficacy and minimal cytotoxicity. At this concentration, cell viability remained consistently high, with values of 96.21%, 102.3%, and 107.3% at 24, 48, and 72 h, respectively, indicating no significant toxicity compared to the control group (p > 0.05). In contrast, higher concentrations (20, 50, and 100 μg/mL) showed a significant reduction in cell viability (p < 0.0001), with viability dropping to 74.21% and 56.3% at 50 and 100 μg/mL, respectively, after 24 h, and further declining to 62.36% and 35.56% after 72 h. These findings suggest that 10 μg/mL of LEA is the optimal concentration, providing effective cellular protection without inducing significant cytotoxicity, making it suitable for further experimental applications.

3.3. Cytotoxicity assessment of restorative materials and LEA-modified formulations

The cell viability of HGFs cultured on NGI, NDC, and LEA-enhanced materials was evaluated over 24, 48, and 72 h using the MTT assay (Fig. 1e). The results demonstrated that the NGI and NDC groups exhibited significantly lower cell viability compared to the control group at all time points (p < 0.0001), with mean viabilities of 55.45% and 60.56% at 24 h, 50.56% and 55.67% at 48 h, and 45.67% and 50.76% at 72 h, respectively. In contrast, the NGI + LEA and NDC + LEA groups showed significantly improved cell viability, with values close to the control group at 24h and 48h and with values exceeding the control group at 72h (p < 0.05). Specifically, the mean viabilities for NGI + LEA and NDC + LEA were 92.76% and 95.67% at 24 h, 104.36% and 105.47% at 48 h, and 107.36% and 108.47% at 72 h, respectively. Viability values marginally above 100% are consistent with reports of antioxidant-treated cultures and likely reflect enhanced mitochondrial metabolic activity rather than supraphysiological proliferation. Statistical analysis using Tukey's multiple comparisons tests confirmed significant differences between the NGI and NDC groups (p < 0.05), with higher cell toxicity in the NGI group.

3.4. Enhanced cell adhesion with LEA-modified restorative materials

The cell adhesion of HGFs to restorative materials (NGI and NDC) and LEA-enhanced materials (NGI + LEA and NDC + LEA) was evaluated over 24, 48, and 72 h (Fig. 2a–c). At 24 h, the NGI and NDC groups exhibited significantly lower cell adhesion compared to the control group (p < 0.0001), with mean adhesion values of 0.55 and 0.60, respectively, while the NGI + LEA and NDC + LEA groups showed improved adhesion, with values of 0.88 and 0.92, respectively, which were not significantly different from the control group (p > 0.05). At 48 h, the NGI + LEA and NDC + LEA groups demonstrated further enhancement in cell adhesion, with mean values of 1.12 and 1.18, respectively. The differences observed at this time point were consistent with those after 24 h of incubation, with the notable exception that the NDC + LEA group exhibited a significant increase in cell adhesion compared to the control group (p > 0.05). By 72 h, the NGI + LEA and NDC + LEA groups reached mean adhesion values of 1.22 (p < 0.01) and 1.28 (p < 0.001), respectively, significantly exceeding the control group and demonstrating a clear improvement over the NGI and NDC groups (p < 0.0001). Statistical analysis using Tukey's multiple comparisons tests confirmed significant differences between the NGI/NDC groups and their LEA-enhanced counterparts at all time points (p < 0.0001), while no significant differences were observed between the NGI and NDC groups at all time points.

Fig. 2.

HGF adhesion to control, NGI, NDC, NGI + LEA, and NDC + LEA surfaces at (a) 1, (b) 2, and (c) 3 days (crystal violet assay). (d) TNF-α and (e) IL-6 secretion after 72 h. (f) ROS and (g) MDA levels after 72 h of culture on test surfaces. Statistical comparisons: $ significantly different from NGI; # significantly different from NDC; ^ significantly different from Control (p < 0.05).

3.5. Regulate inflammatory effects of restorative materials by LEA

The levels of IL-6 and TNF-α cytokines were measured to evaluate the anti-inflammatory potential of NGI, NDC, NGI + LEA, and NDC + LEA groups compared to the unmodified control group. The results revealed that the NGI and NDC groups significantly elevated IL-6 and TNF-α levels compared to the control group (p < 0.001), with mean IL-6 levels of 123.45 pg/mL and 113.56 pg/mL, respectively, and mean TNF-α levels of 96.45 pg/mL and 91.56 pg/mL, respectively. In contrast, the NGI + LEA and NDC + LEA groups demonstrated a remarkable reduction in cytokine levels, with mean IL-6 values of 61.96 pg/mL and 57.07 pg/mL, and mean TNF-α values of 41.96 pg/mL and 37.07 pg/mL, respectively. These values were not significantly different from the control group, indicating that LEA effectively suppressed inflammation. While no significant differences were observed between the NGI and the NDC samples.

3.6. Antioxidative stress effects of LEA-modified restorative materials

The levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) were measured to evaluate the oxidative stress induced by NGI, NDC, NGI + LEA, and NDC + LEA groups compared to the unmodified control group. The results revealed that the NGI and NDC groups significantly elevated ROS and MDA levels compared to the control group (p < 0.001), with mean ROS levels of 150.23 and 140.34, respectively, and mean MDA levels of 4.23 nmol/mg protein and 4.10 nmol/mg protein, respectively. In contrast, the NGI + LEA and NDC + LEA groups demonstrated a remarkable reduction in oxidative stress markers, with mean ROS values of 110.34 and 105.45, and mean MDA values of 2.34 nmol/mg protein and 2.25 nmol/mg protein, respectively. These values were not significantly different from the control group, indicating that LEA effectively mitigated oxidative stress. While no significant differences were observed between the NGI and NDC samples.

4. Discussion

The choice of restorative materials remains critical in dentistry, as these materials must combine mechanical strength, dimensional stability, and long-term biocompatibility [[1], [2], [3]]. While NGIs and NDCs such as Fuji IX GP Extra and Biodentine are widely used due to their favorable handling and bioactive potential, biological challenges including cytotoxicity, oxidative stress, and inflammation continue to limit their clinical longevity [2,4,5,[7], [8], [9]]. Recent reviews on nanocarriers in endodontics highlight promising antimicrobial outcomes but emphasize substantial gaps in pre-clinical validation, reinforcing the need for innovative bioactive nanocarrier-modified dental materials [29]. Encapsulation systems have been shown to markedly improve the stability and biological activity of sensitive biomolecules supporting the concept that nanocarrier protection can enhance functional performance [30]. One innovative approach to addressing these limitations is the use of liposomal carriers to enhance the biological performance of dental materials [14]. Their potential applications include caries and periodontitis treatment, targeted delivery of antibacterial agents, and carriers for biomimetic remineralization [15,16]. Additionally, they can adhere to restorative material surfaces, forming a protective layer that reduces marginal leakage and microbial infiltration [17,18]. The present study demonstrates that incorporating LEA into both NGI and NDC markedly enhances their biological performance, yielding improved cell viability, enhanced adhesion, and suppressed oxidative and inflammatory responses in HGFs.

LEA-modified NGI and NDC restored HGF viability to near or above control levels and significantly improved cell adhesion over 72 h, outperforming unmodified materials. Importantly, LEA treatment reduced intracellular ROS and MDA to baseline levels and suppressed IL-6 and TNF-α secretion, indicating strong antioxidant and anti-inflammatory effects. These outcomes suggest that liposomal delivery of a bioactive polyphenol can directly counteract the adverse cellular effects typically associated with restorative materials. To our knowledge, no prior study has utilized nanoliposomes to incorporate small-molecule polyphenols into dental restorative systems, making this an innovative step toward bioactive, long-lasting restorations.

Our findings are consistent with earlier reports demonstrating the protective effects of polyphenols on dental pulp cells and oral fibroblasts. Lim et al. reported that low concentrations of non-encapsulated polyphenols enhanced collagen cross-linking and promoted proliferation in dental pulp cells [31]. Similarly, Kim et al. observed no cytotoxicity of flavonoids toward osteoblasts at therapeutic doses [32]. However, direct application of polyphenols suffers from low stability, poor water solubility, and rapid degradation in aqueous environments, limitations addressed in our study through liposomal encapsulation [33,34]. By achieving an encapsulation efficiency of 69.7% and a sustained release profile (39% at 24 h, 56% at 72 h), our LEA formulation provides prolonged bioactive exposure at the material interface, likely contributing to the progressive improvements in cell viability and adhesion observed in our assays. Clinical evidence also supports the beneficial impact of nanoparticle-functionalized dental materials, as nanofortified adhesives have been shown to significantly reduce postoperative sensitivity compared with conventional formulations. Such findings reinforce the potential of nano-enabled modifications, whether mechanical or biological, to enhance clinical performance [35]. Similarly, nano-reinforced intracanal medicaments have demonstrated superior clinical outcomes, such as greater reductions in postoperative pain compared with conventional materials, highlighting the growing translational relevance of nanoparticle-enhanced biomaterials [36].

The antioxidant properties of EA are central to its biological effects. EA's polyphenolic structure, with multiple hydroxyl groups, enables efficient scavenging of hydroxyl and superoxide radicals [37,38]. Remarkably, EA can undergo continuous regeneration after neutralizing reactive species, maintaining antioxidant activity even at low concentrations [39]. By reducing ROS levels, LEA may prevent oxidative stress–driven activation of matrix metalloproteinases [40], which degrade ECM components critical for cell adhesion.

In addition to direct radical scavenging, polyphenols have been shown to modulate intracellular signaling pathways. Our observed reduction in IL-6 and TNF-α is consistent with the inhibition of ROS-driven NF-κB activation, a pathway widely implicated in dental material–induced inflammation [19,20]. LEA may also activate the Nrf2 pathway, enhancing the expression of endogenous antioxidant enzymes [27]. Furthermore, EA metabolites, particularly urolithins, can inhibit COX-2 and prostaglandin E2 synthesis [21,22], adding another layer to its anti-inflammatory profile. While these mechanisms provide plausible explanations consistent with the biological trends observed in our study, the present work did not directly evaluate NF-κB, Nrf2, or COX-2 signaling, and therefore these mechanistic interpretations remain hypothetical and warrant confirmation in future molecular analyses.

The liposomal formulation likely amplifies these effects. By providing a cholesterol-stabilized bilayer, the nanoliposomes protect EA from premature degradation, modulate its release, and may directly interact with cell membranes. Electrostatic interactions between the negatively charged EA-containing bilayer and cellular surface receptors could promote integrin-mediated adhesion [24] and enhance ECM deposition [23]. This is supported by our finding that LEA-modified surfaces progressively increased adhesion, surpassing control values by day 3.

Both NGI and NDC face clinical limitations that our approach may address. Fuji IX GP Extra can release residual monomers leading to cytotoxicity [8,41,42], while Biodentine's high pH and ion release can induce inflammation [1,4,5,7]. In our in vitro experiments, LEA-modified surfaces demonstrated reduced oxidative and inflammatory markers compared with unmodified materials, indicating improved short-term biological tolerance under the tested conditions. While these findings raise the possibility that LEA could modulate tissue responses in vivo, such implications require further validation in animal or clinical settings.

The present approach may offer dual potential benefits: (1) biological modulation, reflected by improved cell viability, adhesion, and reduced cytokine levels; and (2) possible structural support, as polyphenols have been reported in other systems to influence ECM-related processes. However, ECM reinforcement or long-term interfacial stability were not directly assessed in this study and remain areas for future investigation. These combined hypotheses could be relevant to high-stress restorations and patient populations with impaired healing, but further mechanistic and functional testing is needed before drawing definitive conclusions.

While our in vitro results are promising, they cannot fully replicate the complex environment of the oral cavity, which includes fluctuating pH, mechanical loading, salivary enzymes, and microbial biofilms. Future work should assess LEA-modified materials under simulated oral conditions, including long-term mechanical and microbiological testing, and in vivo models could further clarify whether the observed biological benefits translate to improved clinical performance. Moreover, mechanistic studies should characterize ECM composition, adhesion protein expression, and potential changes in cell phenotype following exposure to LEA-modified surfaces. Future work will also incorporate SEM or AFM-based characterization to evaluate surface morphology and confirm the uniform distribution of the LEA coating on restorative materials. Another important direction is the use of a broader biological model; future studies will incorporate a dual-cell system including human dental pulp stem cells (HDPSCs) to more comprehensively assess pulpal compatibility and regenerative potential. Additionally, optimization of liposome composition, such as modifying lipid type, surface charge, or ligand functionalization, may further enhance bioactivity and specificity, and potential synergistic effects with other bioactive agents, such as antimicrobial peptides or remineralizing ions, merit investigation.

5. Conclusion

This study presents a preliminary strategy for improving the biological response of NGI and NDC through the surface application of liposomal ellagic acid. Under the in vitro conditions tested, LEA-modified materials showed higher cytocompatibility, improved cell adhesion, and reduced oxidative and inflammatory markers compared with unmodified controls. While these findings suggest that liposomal delivery may offer a promising approach for modulating the biological interface of restorative materials, the underlying molecular mechanisms and clinical relevance were not directly assessed. Future studies incorporating mechanistic analyses, surface characterization, and in vivo models will be essential to fully determine the potential of polyphenol-loaded nanoliposomes in the development of next-generation bioactive restorative systems.

Ethics approval

This work was supported by grant number 654357 from the Research Council of the Faculty of Medicine, Shiraz University of Medical Sciences (SUMS, Iran) with ethic number IR.SUMS.MEDICAL.REC.1401.546. No human participants were directly involved in this study.

Funding

N/A.

CRediT authorship contribution statement

Maryam Rezaeianjam: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing – original draft, Writing – review & editing. Mohammad Hossein Yazdanpanah: Conceptualization, Investigation, Methodology, Validation. Zahra Mohsenpour: Conceptualization, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing. Mahyar Khanlari Goodarzi: Data curation, Formal analysis, Investigation, Methodology, Visualization. Ibragimova Feruza: Data curation, Validation, Writing – review & editing.

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.

Data availability

Data will be made available on request.