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Synergistic effect of iron oxide nanoparticles and ficus carica extract on pectin-PVA electrospun nanofibers for diabetic wound regeneration
†co-authors:
* Corresponding authors: E-mail addresses: 174298670cx@sina.com (X. Chen); zhouxun123@sina.com (X. Zhou)
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Received: ,
Accepted: ,
Abstract
Diabetes mellitus is a widespread metabolic disorder that often leads to chronic, non-healing wounds due to infection, inflammation, impaired extracellular matrix (ECM) formation, and insufficient angiogenesis. This study developed a biocompatible electrospun fiber to promote diabetic wound healing using magnetite iron oxide nanoparticles (MNPs), natural citrus peel pectin, polyvinyl alcohol (PVA), and Ficus carica (FC) fruit extract. The fourier transform infrared (FTIR) spectroscopy analysis was used to understand the interaction of the polymers, Fe₃O₄, and FC extracts in the fiber composite formation. The crystalline nature of the fabricated composite was observed through X-ray diffraction analysis (XRD) analysis. The electrospun fibers displayed a uniform structure, enhanced by a higher PVA content, with an average pore diameter of 30.4 nm and a pore volume of 0.0126 cm3/g, as characterized by a brunauer-emmett-teller (BET) surface analyzer. Morphological characteristics of the fiber were noted, and the variation of the percentage of the Fe₃O₄ influence on fiber formation was examined using scanning electron microscopy (SEM). In vitro analyses, including cell viability, cytotoxicity, scratch wound healing, reactive oxygen species (ROS) staining, and Hoechst staining, were performed on normal and diabetic fibroblast cells. Results demonstrated that the fibers were non-toxic, promoted cell proliferation and migration, reduced oxidative stress, and enhanced wound closure, highlighting their potential in diabetic wound care applications.
Keywords
Diabetes mellitus
Electrospinning
Extracellular matrix
Ficus carica
Nanofiber
1. Introduction
The fabrication of fibrous 3D-scaffolds at the nano or micrometer level for soft tissue engineering has long been encouraged due to its extracellular matrix (ECM) mimicking property. Moreover, the presence of a high degree of interconnecting pores with an appropriate pore size and large surface area enables these fibrous materials to be used as soft tissue engineering frameworks for wound dressings [1,2]. Diabetes mellitus (DM) is a metabolic disorder caused by a dysfunction in glucose metabolism, which leads to the destruction of proteins and lipids, thereby providing a negative effect that favors diabetic complications [3]. About 90% of diabetic cases result from type 2 diabetes mellitus (T2DM) [4]. Diabetic patients are at a high risk of developing chronic diabetic foot ulcers (DFUs), which require prolonged periods of repair and regeneration [5]. DFUs result from an imbalance and damage to small blood vessels and peripheral nerves, which are related to an increase in blood glucose levels [6]. DFUs are challenging to treat, often leading to relapse, and are approximately 15 to 45 times more likely to require amputation than persons without diabetes [7]. Suppressing or lowering blood glucose is a valuable approach to enhance the regeneration of diabetic wounds.
Wound dressing materials are utilized as a defensive barrier against bacterial pathogens during the healing process. To satisfy this condition, these materials ought to be biocompatible and assist with cell connection, expansion, migration, and differentiation for faster wound healing [8]. In 2015, Sadri et al. developed and investigated polymeric nanofibers based on chitosan and polyethylene oxide (PEO) polymers loaded with natural green tea extract on the wound healing effect [9]. They prepared three nanofibers by electrospinning: chitosan, chitosan/PEO, and chitosan/PEO/green tea extract composites. They evaluated the wound healing percentage of these nano-fibrous composites in an in vivo rat model after 16 days. They concluded that their newly developed nanofiber based on chitosan/PEO/green tea extract composites was an ideal wound dressing. Another study based on electrospun nanofibers for application in diabetic wound healing was conducted by Ahmed et al [10]. They prepared nanofibers made from chitosan, polyvinyl alcohol (PVA), and zinc oxide (ZnO) nanoparticles. They evaluated the anti-bacterial and anti-oxidant activity of the chitosan/PVA composite and found these activities increased after adding ZnO. The wound healing property of the chitosan/PVA/ZnO fibrous matrix improved healing over 12 days in an in vivo rabbit model due to the porous nature and fibrous structure of the composite as compared to the chitosan/PVA composite. They concluded that their material would serve as a better platform for treating type-2 diabetic wound infections [10].
Hyperglycemia increases the risk of diabetes and related complications, including myocardial infarctions, nerve damage, stroke, poor vision, limb amputations, and kidney disease [11,12]. Natural bioactive compounds utilized in traditional medicines have been shown to be a valuable choice in avoiding limb amputation [13]. Ficus carica Linn (Ficus carica L.), generally called ‘Figs,’ is one of the members of the genus Ficus belonging to the Moraceae family [14]. Numerous biological activities in health applications of the F. carica plant are the result of the constituents that are present in the form of phenolics, chlorogenic acids, and flavonoids [15,16]. F. carica has been utilized as a traditional medicine in various forms, including Siddha, Ayurveda, and Unani [17]. The health benefits of Ficus include diabetes (endocrine system), liver diseases, asthma and cough (respiratory system), ulcers (gastrointestinal system), and infectious skin diseases [18,19]. In particular, Ficus fruits are generally used as a nutrient food, and the presence of polysaccharides and polyphenols in the fruit makes it ideal to be utilized as a medicine [20]. In 2018, Mopuri et al. showed that the ethanolic extract of figs has higher polyphenols and flavonoids (104.67 ± 5.51 μg/mL and 81.67 ± 4.00 μg/mL) content compared with other organic and inorganic solvents, and the inhibition of α-amylase and α-glucosidase confirmed its anti-diabetic activity [18]. Previous research reports confirm that figs contain bioactive compounds with anti-diabetic and immunomodulatory effects, as well as anti-inflammatory properties [19]. F. carica is well established for its anti-oxidant properties, and its activity has been further enhanced by the use of iron oxide (magnetite) nanoparticles for its high surface area and anti-inflammatory properties.
Pectin is a natural polysaccharide found in various fruits and vegetables and is a source of soluble dietary fiber, which has a significant anti-diabetic effect [21]. Moreover, pectin is safe for human consumption, including food products and pharmaceutical products [22]. In a previous study, Palou et al. (2015) demonstrated that the supplementation of 10% apple pectin in adult rats for one month had the ability to counteract metabolic disorders due to age-related factors, including obesity, accumulation of fat and the resistance of peripheral insulin and leptin. It was concluded that the uptake of pectin facilitates metabolic health [23]. Liu et al. (2016) elucidated the anti-diabetic role of citrus pectin in type 2 diabetic rats. They observed that glucose levels were significantly reduced after 4 weeks of administration of citrus pectin. They concluded that this anti-diabetic effect might occur through the regulation of phosphatidylinositol 3 kinase(PI3K) /protein kinase B (AKT) signaling pathways [24].
To enhance diabetic wound healing, this study introduces a novel multifunctional electrospun nanofibrous composite that incorporates Ficus carica (fig) extract and magnetite (Fe₃O₄) nanoparticles into a pectin–PVA matrix. Unlike traditional wound dressings, this composite combines the antibacterial and magnetic responsiveness of Fe₃O₄, the biodegradable and bioadhesive properties of citrus-derived pectin, and the anti-inflammatory and anti-diabetic bioactivities of Ficus carica. While the fig extract promotes wound regeneration through pro-healing and antioxidative effects, the addition of just 2% Fe₃O₄ ensures better biocompatibility without compromising fiber structure. This unique blend of materials offers a synergistic therapeutic platform that addresses the complex challenges of chronic diabetes wounds, including inadequate ECM formation, delayed angiogenesis, and oxidative stress. To our knowledge, this is the first study to develop a PVA/pectin nanofibrous scaffold loaded with Ficus carica and Fe₃O₄ nanoparticles for diabetic wound healing. It presents a promising option for next-generation bioactive wound dressings.
2. Materials and Methods
2.1. Materials
Ficus carica fruit was purchased from the local market in Madurai, Tamil Nadu, India. The average molecular weight of 160,000 for PVA (89% hydrolyzed), ethanol, liquid ammonia, ferric chloride hexahydrate (FeCl3·6H2O), and ferrous sulfate heptahydrate (FeSO4·7H2O) was obtained from Sigma-Aldrich. Pectin was extracted from citrus peel. Double-distilled water (DDW) was used in all the experiments.
2.2. Ficus carica fruit extract preparation
The ethanolic extract of Ficus carica was prepared using a previously reported procedure with slight modifications [18]. Fresh fruits were washed twice with DDW and ethanol, then ground into a paste. A total of 100 g of fruit paste was extracted in 500 mL of ethanol for 60 h. The resulting solution was filtered through Whatman filter paper, and then the ethanol was evaporated at 50°C under reduced pressure using a rotary evaporator. The FC isolation was then kept at 4°C for future use.
2.3. Preparation of Fe3O4 (Magnetite) nanoparticles
Magnetite nanoparticles (MNPs) were synthesized via the chemical co-precipitation method following a previously reported protocol with minor modifications [25]. Ferrous (Fe2⁺) and ferric (Fe3⁺) ions were used in a 1:2 molar ratio in an ammonia solution. First, 0.03 M FeSO₄·7H₂O and 0.06 M FeCl₃·6H₂O were combined and dissolved in 200 mL of DDW while being continuously stirred at 80°C in a nitrogen environment. After that, 12 mL of a 25% ammonia solution was added dropwise while being constantly stirred until the pH reached about 10–11, at which point a black precipitate that indicated the development of magnetite was formed. The reaction mixture was maintained at 80°C for an additional 30 min to ensure complete precipitation. To get rid of extra ions and leftover ammonia, the resulting MNPs were separated using centrifugation (or magnetic decantation) and repeatedly cleaned with double-distilled water until a neutral pH was reached. The refined magnetite nanoparticles were then kept for later use after being dried at 60°C.
2.4. Preparation of Pec-PVA, Pec-PVA/Fe3O4, and Pec-PVA/Fe3O4/Ficus carica extract solutions for electrospinning
Electrospinning solutions were prepared by adding different weight percentages (2% and 20%) of Fe3O4 solution into 20 mL of Pec-PVA polymer solution under vigorous stirring at 25°C for three hours. Based on initial optimization investigations, Fe₃O₄ values of 2% and 20% were chosen to represent low and high nanoparticle loading limits that preserved homogeneous nanoparticle dispersion, uniform fiber shape, and reliable electrospinning. The Pec-PVA polymer solution was prepared by adding a 2% aqueous solution of pectin dropwise into a 30% aqueous PVA solution at 80°C under continuous stirring for three hours. The Ficus carica extract (0.25 g in 5 mL of water) was then slowly mixed into the Fe3O4-loaded polymeric solution. It stirred for 18 h at 25°C to obtain a homogeneous reaction mixture, which was then loaded into a syringe for electrospinning. Solutions of Pec-PVA and Pec-PVA/Fe3O4 were prepared using the same procedure, omitting the addition of Fe3O4 and Ficus carica extract, respectively.
2.5. Fiber formulation via electrospinning
The reaction mixture was individually loaded into 5 mL syringes equipped with a 21-gauge precision needle, which was connected to a high-voltage power supply set at 22 kV. A rotating drum collector covered with aluminum foil was used, with a flow rate set at 0.75 mL/h and a drum speed of 10 rpm. The syringe needle was kept 15 cm away from the drum collector. The collected fiber samples were dried overnight on the aluminum foil at 60°C to ensure complete solvent evaporation [9]. The fibrous composites prepared using the electrospinning method, including PVA, Pec-30% PVA, Pec-30% PVA/Fe3O4, and Pec-30% PVA/2% Fe3O4/FC extract composites, are presented in Figure 1.
- Prepared fibrous composites from the electrospinning method. (a) PVA, (b) Pec-30% PVA (c) Pec-30% PVA/Fe3O4 and (d) Pec-30% PVA/2% Fe3O4/FC extract composites.
2.6. Physicochemical characterizations
2.6.1. Functional group analysis
The presence of functional groups and the formation of target composites were examined using a Nicolet 380 Fourier transform infrared (FT-IR) spectrometer (Thermo Fisher Scientific). The analysis was conducted in the 4000-400 cm-1 range with the KBr pellet technique at a resolution of 4 cm-1.
2.6.2. X-ray diffraction analysis
The crystallinity and phase identification of composite samples were characterized using a Bruker D8 Endeavor X-ray diffraction instrument, with scanning angles from 20 to 60° and a scanning rate of 0.02° in 2θ. The instrument operated at 40 kV and 30 mA with Cu Kα incident radiation.
2.6.3. Morphological analysis
2.6.3.1. Scanning electron microscopy analysis
The sample’s surface morphology and elemental composition were examined using a scanning electron microscopy (SEM) system equipped with a Noran energy dispersive X-ray (EDX) analyzer (JEOL JSM-6400, Japan). To enhance conductivity, the samples were coated onto glass plates and subjected to ion sputtering before analysis.
2.6.3.2. Porosity and surface area measurements
The Barrett−Joyner−Halenda (BJH) and Brunauer-Emmett-Teller (BET) methods were employed to measure pore size and surface area, respectively, using a Tel Micro Tract analyzer (Belcork, Japan) under continuous adsorption at 77 K. Prior to nitrogen adsorption and desorption analysis, all samples were degassed at 100°C.
2.7. Antimicrobial activity studies
2.7.1. Minimal inhibitory concentration (MIC) of the fibrous composite
The antibacterial activity of the fibrous composites was evaluated using the agar well diffusion (growth inhibition zone) assay against Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922), obtained from the American Type Culture Collection (ATCC, Virginia, USA). The bacterial strains were maintained as glycerol stocks at −80 °C and subcultured in nutrient broth prior to experimentation. Fresh bacterial suspensions were prepared by inoculating the cultures into nutrient broth and incubating at 37 °C for 18 h. The bacterial concentration was adjusted to approximately 10⁶ CFU/mL. Sterile MuellerHinton agar plates were uniformly swabbed with the bacterial suspension using a sterile cotton swab to ensure even lawn formation [26]. Wells of 6 mm diameter were aseptically punched into the agar using a sterile cork borer. The fibrous composite samples, namely FC, Fe₃O₄, Pectin, Pec-PVA, Pec-PVA/Fe₃O₄, and Pec-PVA/Fe₃O₄/FC, were dispersed in sterile physiological saline, and a fixed concentration of 1.5 mg/mL was used for all samples. A defined volume of each sample suspension was carefully introduced into the respective wells. Plates were then incubated at 37 °C for 24 h under aerobic conditions. After incubation, the diameter of the clear inhibition zone (mm) around each well was measured using a digital Vernier caliper. All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation. The quantitatively measured inhibition zones are presented in Supplementary Figure S1, illustrating the comparative antibacterial performance of the fibrous composites against both Gram-positive and Gram-negative bacterial strains.
2.8. In-vitro studies
2.8.1. Cell culture
This study utilized WS1 human skin fibroblast cells (ATCC® CRL-1502™). These cells were cultured in minimum essential medium (MEM) supplemented with 1 mM sodium pyruvate, 0.1 mM non-essential amino acids (NEAA), 2 mM L-glutamine, 1 mM amphotericin B, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS). To create a diabetic cell model, WS1 cells were cultured for at least 14 days in complete medium containing an additional 17 mM/L D-glucose, achieving a total glucose concentration of 22.6 mM/L [27,28]. The cells were maintained in 3.4 cm diameter dishes within a CO2 incubator at 37°C, after which they were wounded as described below.
2.8.2. Trypan blue assay method for cell viability analysis
Biocompatibility testing was conducted using three composite fibers: Pectin-PVA/20% Fe3O4, Pectin-PVA/2% Fe3O4, and Pectin-PVA/2% Fe3O4/FC. The sterilized composites at varying concentrations (10, 25, 50, 75, and 100 μg/mL) were seeded with WS1 cells. The trypan blue exclusion assay was used to assess the cell viability, where viable cells with intact membranes remained unstained. In contrast, non-viable cells with compromised membranes absorbed the dye and appeared blue. Using the Invitrogen Countess™ II FL Automated Cell Counter, the viable cell count was determined [29].
2.8.3. Adenosine triphosphate assay (ATP) assay for measurement of metabolic viability
Cellular ATP levels were measured with the CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7571) according to the manufacturer’s instructions. Briefly, 50 μL of cell lysate was mixed with an equal amount of ATP assay reagent, incubated in the dark at 25°C for 10 min, and then luminescence was measured using a Perkin-Elmer Victor3 multi-plate reader.
2.8.4. Lactate dehydrogenase (LDH) assay for cytotoxicity
Cytotoxicity of the composites was assessed by measurement the LDH concentration using the Cytotox 96® Non-Radioactive Cytotoxicity method (Promega, G1780). WS1 cells were cultured on Pectin-PVA/20% Fe3O4, Pectin-PVA/2% Fe3O4, and Pectin-PVA/2% Fe3O4/FC at various concentrations (10–100 μg/mL). After incubation, 50 μL of supernatant was mixed with 50 μL of reconstituted substrate mix and incubated in the dark for 30 min. Optical density was measured at 490 nm using a Perkin-Elmer Victor3 multi-plate reader [30].
2.8.5. In vitro scratch wound assay and cell migration analysis
WS1 cells (6 × 10^5 cells per well) were seeded in six-well plates and allowed to grow until they reached 80% confluence. A scratch wound was made using a sterile P200 pipette tip. After creating the scratch, the cells were incubated in a CO2 incubator for 30 min, washed twice with phosphate buffered saline (PBS), and then cultured in fresh medium with treatment composites for 48 h. Cell migration was monitored, and images were captured every 12 h using an Olympus inverted light microscope until wound closure.
2.8.6. Hoechst 33258 staining
The morphology of wounded and diabetic cells cultured on different composites was assessed using Hoechst 33258 staining. After 48 h of cultivation, cells were washed thrice with PBS and stained with Hoechst 33258 (10 μg/mL) for 7 min at 37°C in a 5% CO2 atmosphere. Excess dye was removed by washing with PBS, and cells were visualized using a Carl Zeiss Axio Observer Z1 microscope (352Ex/461Em).
2.8.7. Reactive oxygen species (ROS) staining in intracellular analysis
Intracellular ROS generation in WS1 cells was evaluated using dihydroethidium (DHE) staining. Cells (6 × 10⁵) were seeded on sterile coverslips and cultured for 12 h, followed by treatment with different samples for 48 h. After treatment, cells were incubated with DHE (10 μM) for 30 min at 37 °C in the dark. The cells were then washed twice with PBS and counterstained with DAPI (4’,6-diamidino-2-phenylindole) (1 μg/mL) for 5 min at room temperature. Fluorescence images were captured using a Carl Zeiss Axio Observer Z1 fluorescence microscope. DHE fluorescence was detected using excitation/emission wavelengths of 535/610 nm (red fluorescence), while DAPI-stained nuclei were observed using excitation/emission wavelengths of 352/461 nm (blue fluorescence).
2.9. Statistical analysis
Experimental data (n = 3) are shown as mean ± standard error (SE). Each biological replicate was measured twice per assay, and the average was used for analysis. Statistical analysis was conducted using analysis of variance (ANOVA), followed by Tukey’s multiple comparison test (Prism, version 5.0). Significance was defined as P < 0.05.
3. Results and Discussion
3.1. FT-IR analysis
The prepared composite fibers’ functionality was evaluated using FT-IR analysis, with the results depicted in Figure 2. The functional characterization of the Pectin and PVA polymeric fibers and their corresponding FTIR spectra is shown in Figure 2a. This spectrum provides insight into the structural functionality changes and the major interactions involved in the formation of Pectin and PVA fibers. The broad spectrum ranging from 3275 to 3290 cm-1 resembles the hydroxyl (-OH) groups stretching vibrations in both polymers. The two strong bands at 2906 and 2943 cm-1 represent C-H stretching vibrations, while the band at 1718 cm-1 designates stretching vibrations of carbonyl (C=O) from the carboxy (COO-) group of pectin. Two functional groups, specifically C=C and -COO from the carboxy group, produce a band at 1650 cm-1. The peaks appearing at 1373 and 1428 cm-1 are related to the deformation vibrations of C-H and CH2, respectively. Moreover, the band at 1244 cm-1 relates to the C-O-C stretching vibration of the 1-4 linked D-galacturonic acid in the pectin molecule [22]. The spectrum presented in Figure 2b illustrates the functionality of the synthesized MNPs. In this spectrum, two well-defined bands at 561 and 634 cm-1 confirm the presence of an iron-oxygen bond (Fe-O). The strong band at 1638 cm-1 and the broad band between 3436 and 3491 cm-1 are attributed to the bending vibrations of absorbed water molecules and a surface hydroxyl (-OH) group, respectively. This spectral information clearly indicates the formation of Fe3O4 MNPs. The composite formed from Pec-PVA/2% Fe3O4 is evident in Figure 2c. All the vibrational bands from the spectrum of Pec-PVA in Figure 2a and the MNPs spectrum in Figure 2b are present in Figure 2c with slight deviations in wave numbers, confirming the formation of Fe3O4 embbeded Pec-PVA polymeric fibers. The confirmation of the final composite of ficus carica fruit extract-loaded Pec-PVA/Fe3O4 fibers was characterized for their functional groups and is shown in Figure 2d. The aforementioned Pec-PVA/Fe3O4 bands, along with some additional vibrational stretching frequencies, are observed in Figure 2d. The strong band at 1041 cm-1 corresponds to the C-O vibrational frequency of the fruit extract. The band at 2149 cm-1 corresponds to the alkene (C=C) functional group, and the broad band observed around 3035-3177 cm-1 relates to the phenolic -OH group for principal molecules like phenolic acids, flavonoids, and anthocyanins [31]. This observation shows that the FC extract was successfully loaded onto Pec-PVA/Fe3O4, resulting in the composite fibers of Pec-PVA/Fe3O4/FC extract [17]. These FT-IR results reveal significant interactions among the components in the fibrous matrix and FC extracts, with the additional peaks arising from the presence of FC extract within the fibrous matrix.
- FT-IR spectrum of a) Pec-PVA, b) Fe3O4, c) Pec-PVA/Fe3O4, and d) Pec-PVA/Fe3O4/FC; and XRD patterns of e) Pec-PVA, f) Pec-PVA/Fe3O, and g) Pec-PVA/Fe3O4/FC composites.
3.2. XRD analysis
The crystallization of the prepared electrospun composite fibers was evaluated using XRD analysis, and the diffraction pattern is presented in Figure 2b. The post-spinning of the Pec-PVA composite fibers resulted in the formation of four primary diffraction peaks noted at 2θ value of 15.64, 19.8, 25.04, and 28.4 degrees, corresponding to the 001, 101, 200, and 201 planes, respectively, as shown in Figure 2e [19]. These planes of the Pec-PVA composite experience slight shifts from their original positions due to the influence of MNPs on their polymeric chains. The diffraction patterns of Pec-PVA/Fe3O4 shown in Figure 2f include additional peaks along with the pattern of Pec-PVA fiber, specifically at 31.72, 35.27, 43.61, 57.39, and 63.03 degrees, which correspond to the “hkl” Miller indices of the 220, 311, 400, 511, and 440 planes respectively [32]. The intensity and the position of these particular planes of MNPs are decreased from their intensity of the purest, un-conjugated form due to the polymeric scaffold that strongly influences their crystalline nature [32]. As a result, the Pec-PVA/Fe3O4 composite exhibits crystallinity along with an increased amorphous nature. After incorporating FC extract into the polymeric-MNPs composite as Pec-PVA/Fe3O4/FC fiber, no new diffraction peaks were detected, nor were there any significant changes in their crystalline structure compared to the earlier composite of Pec-PVA/Fe3O4 fibers, as shown in Figure 2g.
3.3. Surface morphology observation of fiber composite
PVA was added in various percentages to optimize the fiber formation by pectin: 10%, 20%, and 30%. The pure pectin and the pectin fibers with 10%, 20%, and 30% PVA were characterized for their surface morphology using the SEM technique. The pure pectin solution does not produce long fibrous structures but instead forms short spherical fibers, as shown in Figure 3(a), due to the non-conducting nature of the pectin molecule, which prevents the formation of linear fiber structures. Therefore, 10% PVA was added to increase pectin’s spinnability, which creates non-uniform fiber networks containing spherical pectin particles, as indicated by the blue arrows (Figure 3b). Figure 3c shows the Pec-PVA composite with increasing percentages of PVA from 10% to 30%, resulting in a uniform structure of interconnected fibers with tiny pores. Additionally, Fe3O4 MNPS was added to the P-PVA fiber and optimized with two different percentages of Fe3O4 MNPS: 2% and 20%. After incorporating Fe3O4 into the Pec-PVA composite, small white dots (denoted by the black arrows) within the fiber network are visible (see Figures 3d and Figures 3e for 2% and 20% Fe3O4, respectively). The addition of 20% Fe3O4 affected the fiber structure formation, as the 2% Fe3O4 embedded P-PVA fiber was then used for loading the FC fruit extract. After loading the FC extract, the quality of the unique compact fiber with pores was enhanced (see Figure 3f). This result provides strong support for the electrospinning process with the FC principal compound extract, conducted at a voltage of 22 V and a flow rate of 0.75 mL/h to produce a unique and compact porous fiber.
- SEM morphology of a) pectin solution, b) Pec-10% PVA, c) Pec/ 30% PVA, d) Pec/ 30% PVA/ 20% Fe3O4, e) Pec/ 30% PVA/ 2% Fe3O4, and f) Pec/ 30% PVA/ 2% Fe3O4/ FC extract. (Blue arrows indicate the presence of pectin particles, and black arrows indicate the presence of MNPs in the fibrous networks).
3.4. Surface area and pore volume analysis
The Barrett-Joyner-Halenda (BJH) and Brunauer-Emmett-Teller (BET) methodologies were applied to determine the surface area, pore volume, and diameter of the prepared materials. Figure 4(a) displays the adsorption and desorption isotherms, while Figure 4(b) presents the pore volume distribution of the Pec/PVA/Fe3O4/FC fiber. According to the BJH studies, the Pec/PVA/Fe3O4/FC fiber showed a surface area of 2.034 m2/g. This fiber’s average pore volume and pore diameter are reported as 0.0126 cm3/g and 30.4 nm, respectively (Figure 4b). The pore size distribution graph illustrates that most of the pronounced pore peaks are found in the mesopores range (2-50 nm), indicating that mesopores are more prevalent than micropores. The values for surface area and pore volume obtained are comparable to those reported for other polymeric and composite-based fibrous materials in recent research. A recent study by Zhang et al. (2023) on Fe3O4-incorporated PVA-based fibers found a surface area ranging from 1.8 to 3.5 m2/g, which is consistent with our findings [33]. Similarly, the nanostructured polymer-iron oxide hybrid materials indicated pore diameters ranging from 25 to 40 nm, further supporting the observation that mesoporosity is a predominant characteristic of such composite fibers [34].
- (a) N2 adsorption and desorption isotherms at 77 K and (b) pore size distribution of Pec/ PVA/ Fe3O4/ FC fiber.
3.5. In-vitro analysis
3.5.1. Cell viability and cytotoxicity
The biocompatibility of the prepared composites at different concentrations was assessed by measuring WS1 cell viability. WS1 cells were cultured in 3.4 cm diameter dishes. Once they reached 80% confluency, various treatment solutions, including Pectin-PVA/ 20%Fe3O4, Pectin-PVA/2%Fe3O4, and Pectin-PVA/2%Fe3O4/FC fibrous composites at concentrations of 10, 25, 50, 75, and 100 μg/mL, were added and incubated for 48 hrs. A higher level of cell viability was observed in cells incubated with Ficus carica fruit extract-loaded fibers compared to the control (Figure 5a). Furthermore, at a concentration of 75 μg/mL, composite fibers made from Pectin-PVA/2%Fe3O4/FC exhibited a significant increase in cell viability (95.2%). In all the composites at every tested concentration, the viability of WS1 cells remained above 85%. This could be attributed to the formation of fibrous networks that act as an ECM1. The ATP luminescent assay also assessed the viability of normal cells. This assay is based on the principle that the light emitted during luciferin formation is directly proportional to ATP concentration, with results expressed as relative luminescence units (RLU). As depicted in Figure 5(b), the ATP luminescent assay results align closely with those of the Trypan blue exclusion assay.
- Cell viability of fibrous composites composed of Pectin-PVA/20% Fe₃O₄, Pectin-PVA/2% Fe₃O₄, and Pectin-PVA/2% Fe₃O₄/FC at various concentrations using the following methods: (a) Trypan blue exclusion assay, (b) ATP luminescence assay, and (c) LDH cytotoxicity assay. Data are presented as mean ± SEM (n = 3). Statistical significance is indicated as *P ≤ 0.05 compared to Pectin-PVA/20% Fe₃O₄ at the same concentration, and *P ≤ 0.05 compared to the control group.
Additionally, the release of lactate dehydrogenase (LDH) was measured to assess the cytotoxicity of the synthesized composites [35]. Following treatment with 100 μg/mL of the composite, LDH levels showed a slight increase compared to the control cells, which meant 100 μg/mL slightly induced a toxicity effect. However, there was no significant difference compared to other concentrations, except for 75 μg/mL (Figure 5c). At this concentration (75 μg/mL), the release profile of LDH was significantly decreased over the control. These results strongly reveal that the composites at a concentration of 75 μg/mL have a positive effect of protection against cell damage. In particular, the composite Pectin-PVA/ 2% Fe3O4/FC fiber exhibited a good protective effect on normal fibroblast cells. This effect may be due to the anti-inflammatory effect of both FC and MNPs present in the fibrous composites.
All three in-vitro assays revealed that WS1 cells remained viable with a higher viability rate, indicating that our prepared fibrous composite was not cytotoxic in nature. From these results, it was clear that the addition of MNPs and FC extract to the Pectin-PVA matrix did not produce any toxic effect on cell viability due to the biocompatible nature of polymers with FC extract and the anti-inflammatory properties of the MNPs. Notably, results were better when treating the cells with 75 μg/mL of the composite fibers; however, viability decreased when the concentration exceeded this level, indicating that the biocompatibility of the fabricated biocomposite depended on the concentration. This composite fiber may also serve as an ECM for wound regeneration in in vivo applications, as reported with other fibrous composites [9,10].
3.5.2. In vitro scratch wound and morphology changes
Cellular morphology in various composites at different concentrations was analyzed qualitatively by comparing the cell morphology at 48 hrs. In normal (Figure S2) and diabetic (Figure 6) cell models, cells preserved their characteristic spindle shape, with negligible detachment after the culture dish or cell rounding, indicating cellular stress or death. As shown in Figure S2, at a concentration of 75 μg/mL of Pectin-PVA/2% Fe3O4/FC fibrous composite, normal cells were more confluent than those in other composites at various concentrations (25, 50, 75, and 100 μg/mL). The same effect of Ficus carica extract-loaded fibers was observed in diabetic cells (Figure 6). Likewise, the composite of Pectin-PVA/2% Fe3O4/FC fiber at 75 μg/mL resulted in increased cellular proliferation of diabetic cells.
- Optical morphology images of diabetic cell growth on different composites at different concentrations for 48 h (Magnification = x100).
Figure 7 shows the morphology of normal and diabetic wounded cells. At 0 h, the wound margins of both kinds of cells treated with Pectin-PVA/2% Fe3O4/FC fiber composites were well-defined. After this, the wound margins became less distinct, and cells were observed migrating into the “wound” or central scratch (as indicated by arrows). At 48 h, migration occurred at a faster rate following Pectin-PVA/2% Fe3O4/FC treatment, as evidenced by the increased presence of fibroblast cells in the center of the scratch for both normal and diabetic wounded cells models18.
- Optical morphology images of wounded and diabetic wounded cells. Control cells were not treated with composite, while experimental models were treated with 75 μg/mL Pectin-PVA/2% Fe3O4/ FC fibrous composite for 0, 12, 24, and 48 h. The yellow arrow indicates the central scratch (‘wound’) between the two wound margins.
3.5.3. Hoechst 33258 staining to assess the nuclear damage
Hoechst 33258 stained the wounded and diabetic cells after treatment with Pectin-PVA/2% Fe3O4/FC fiber composites, as presented in Figure 8. As shown in Figure 8, the composite demonstrates favorable cell growth over time. After 48 h, number of live nuclei was identified in both wounded and diabetic cells treated with the composites compared to their control. The pectin-PVA/2% Fe3O4/FC fiber composite-treated cells showed uniformly stained nuclei, indicating their dense, spherical shapes without any visible damage.
- Fluorescence microscopy images showing nuclear morphology of wounded and diabetic wounded cells stained with Hoechst 33258 before treatment (control) and after treatment with Pectin–PVA/2% Fe3O4/FC fibrous composite at 0, 12, 24, and 48 h. Wounded control cells (a–d); wounded treated cells (e–h); diabetic wounded control cells (i–l); diabetic wounded treated cells (m–p).
3.5.4. ROS staining
Figure 9 illustrates that the wounded and diabetic wounded cells were stained with ROS. The blue dye specifies DAPI staining Figure 9 (a-d), although the red represents ROS staining Figure 9 (e-h). The cell nucleus appeared circular. No apoptosis was noted in either the wounded or diabetic wounded cells following treatment with Pectin-PVA/2% Fe3O4/FC fibrous composites incubated for 48 h. In both the wounded and diabetic wounded cells, the presence of ROS was significantly decreased after 48 h of treatment compared to the control. In the diabetic wound control, a greater number of cells exhibited the presence of ROS.
- DAPI-stained nuclei (a-d), ROS staining (e-h), and merged images of DAPI and ROS staining (i-l) of wounded and diabetic wounded cells were observed before treatment (control) and after 48 hrs of treatment with the Pectin-PVA/2% Fe₃O₄/FC fibrous composite (Images were captured at a magnification of 200x).
The positive results in the biocompatibility of the various composites arise from their fibrous nature. The presence of Fe₃O₄ nanoparticles enhances cell viability in both normal and diabetic wounded cells without causing cytotoxicity and supports wound closure. The formation of a mesoporous fibrous network promotes fibroblast cell viability. Overall, the Pec-PVA/2% Fe3O4 fibrous composite is expected to be used as a wound dressing in future clinical applications, and the individual composite can serve as a wound dressing for both regular and diabetic wounds.
4. Conclusions
In this investigation, we successfully fabricated a new fibrous network composed of magnetite nanoparticles and FC extracts incorporated into a pectin-polyvinyl alcohol polymeric fiber through electrospinning to accelerate delayed wound healing in diabetes. SEM morphology of the fibrous structure indicated that the percentage of Fe3O4 highly influenced the formation of fibers in the electrospinning process. The 20% of Fe3O4 loaded fiber increased irregularities due to the agglomeration of the magnetite nanoparticles, which limits the spinnability of the composite, and 2% of Fe3O4 loaded fiber observed a smooth fiberous structure. The FT-IR results reveal significant interactions among the components in the fibrous matrix, Fe₃O₄ nanoparticles and FC extracts in the fiber formation. Pec-PVA/Fe3O4/FC fiber was crystalline along with an increased amorphous nature from the XRD determination, it will provide bioactivity needed for the cellular interactions and sustainable wound regeneration. The cell viability of the PVA/2% Fe3O4/FC fiber composite showed a higher viability rate in all three in-vitro assays, indicating that the fabricated fibrous composite was not cytotoxic in nature. Cell migration studies revealed a faster rate under the Pectin-PVA/2% Fe3O4/FC fiber composite treatment. The pectin-PVA/2% Fe3O4/FC fiber composite-treated cells showed uniformly stained nuclei with dense, spherical shapes without any visible damage. Overall, pectin-PVA/2% Fe3O4/FC fiber composite demonstrates a promising strategy for fabricating bioactive, magnetically responsive fibrous wound dressing materials with biocompatible materials with a scalable electrospinning approach. The fabricated composite provides a significant therapeutic dressing material for diabetic wound management in the near future.
Acknowledgment
The authors thank the Chongqing Bureau of Science and Technology (CSTB2023JXJL-YFX0028) for research funding to carry out the research work.
CRediT authorship contribution statement
YZ: writing–review, data curation, formal analysis, investigation, methodology, visualization, writing–original draft; YL: investigation, methodology, resources, validation, writing–original draft; QF: investigation, methodology, resources, validation, writing–original draft; MR: methodology, resources, validation, writing–original draft; YL: conceptualization, funding acquisition, investigation, methodology, project administration, writing–original draft, writing–review and editing; XZ: conceptualization, funding acquisition, investigation, methodology, project administration, writing–original draft, writing–review and editing.
Declaration of competing interest
There are no conflicts of interest.
Data availability
All data generated or analyzed during this study are included in this published article. Additional datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Declaration of generative AI and AI-assisted technologies in the writing process
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Supplementary data
Supplementary material to this article can be found online at https://dx.doi.org/10.25259/AJC_905_2025.
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